WO2019022187A1 - 抗cd147抗体 - Google Patents
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- WO2019022187A1 WO2019022187A1 PCT/JP2018/028047 JP2018028047W WO2019022187A1 WO 2019022187 A1 WO2019022187 A1 WO 2019022187A1 JP 2018028047 W JP2018028047 W JP 2018028047W WO 2019022187 A1 WO2019022187 A1 WO 2019022187A1
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
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- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C12N5/10—Cells modified by introduction of foreign genetic material
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C07K2317/622—Single chain antibody (scFv)
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- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/734—Complement-dependent cytotoxicity [CDC]
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- C07K2317/75—Agonist effect on antigen
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates to an anti-CD147 antibody exhibiting excellent antitumor effect, a method for producing the anti-CD147 antibody, an antitumor agent containing the anti-CD147 antibody and the like.
- CTLA4 antibody and PD1 antibody developed as antibody drugs showing excellent drug stability and specificity have high response rate to melanoma and some solid cancers by activation of T cells and other immune cells. In some cases it cures and is the gospel for cancer patients.
- Treatment with these therapeutic agents has been attempted for many refractory solid cancers, but in pancreatic cancer, liver cancer, etc., most cancers show no sensitivity to the drug, and removal by surgery or conventional methods Even in the treatment based on anticancer drugs, recurrence is recognized at a high rate, and there is a need for a therapeutic method and a therapeutic agent that bring about radical cure.
- CD147 is a single-pass transmembrane protein with 2 to 3 immunoglobulin-like domains and interacts with CD147, such as CD44, Integrin family molecules, CD98, VEGFR, CypA / B, MCT1 / 3/4 proliferation, invasion, By interacting with molecules on the extracellular or cell membrane surface involved in inflammation, downstream signal related molecules, FAK, MEK, Erk, JAK / STAT, AKT, MAPK family molecules are activated, and MMP and other proteases are produced It is known to promote cancer growth, metastasis, and invasion. In addition, in patients with liver cancer and pancreatic cancer mentioned above, it is reported that the survival period is short and the prognosis is poor when the expression of CD147 is high, and it is considered as one of cancer treatment target molecules.
- ABX-CBL As antibodies targeting CD147, ABX-CBL and Licartin have been clinically administered to humans. ABX-CBL not only inhibits the binding of CD147 to cyclophilin A and suppresses the activity of T cells, but also kills normal cells including CD147 positive T cells in a complement-dependent manner in blood. It shows activity. Although clinical trials have been advanced for GVHD as a target disease, drug effects are insufficient, severe muscle pain is observed, and approval as a drug has not been achieved (Patent Document 1, Non-patent Document 1).
- Licartin is a biological preparation in which the radioactive isotope element iodine 131 is added to the Fab'2 part of the HAb 18 antibody, and it has been approved in China for liver cancer as an applicable cancer type (Non-patent document 2, Non-patent document 3) . Licartin lacks the Fc portion of antibodies that activate immune cells and complement, and although there are no reports of immune-mediated toxicity, there are no reports of eradicating liver cancer in clinical practice.
- CD147-targeted antibodies include anti-CD147 monoclonal antibody (patent document 2) that blocks angiogenesis, biological activity associated with CD147 such as VEGF-producing matrix metalloproteinase, and the like, and inhibits T cell activation.
- Anti-CD147 monoclonal antibody Non-patent document 4
- an antibody specifically binding to a CD147 molecule characterized by having ADCC activity and CDC activity (patent document 3) are known.
- CD147 antibody which has no effector function and exhibits an antitumor effect.
- the object of the present invention is to provide a novel anti-CD147 antibody having a novel pharmacological action, an excellent safety, and a high anti-tumor effect, a pharmaceutical comprising the antibody, and a treatment of a tumor using the antibody or the pharmaceutical. It is to provide a method etc.
- the present inventors diligently studied to achieve the above-mentioned problems, and for the first time found that activation of related signal molecules via CD147 is associated with an antitumor effect. Then, the present inventors activated CD147 and succeeded in obtaining a CD147-specific antibody showing a high antitumor effect.
- the antibody of the present invention is characterized in that it exhibits a high anti-tumor effect independently of the effector function. Also, although antibodies showing anti-tumor effects depending on effector function have been reported so far, the antibody of the present invention does not act on T cells and PBMCs, and does not depend on effector function. It is an excellent antibody that has high anti-tumor effects and is expected to be safe as a pharmaceutical.
- the antibody of the present invention exhibits significantly higher efficacy in liver cancer cells than sorafenib, which is used as one of the standard therapeutic agents for liver cancer.
- the antibodies of the present invention exhibit significantly greater efficacy in pancreatic cancer cells than gemcitabine, which is used as one of the standard treatments for pancreatic cancer.
- the antibody of the present invention exhibits significantly higher efficacy in chronic myelogenous leukemia cells than imatinib, which is used as one of the standard therapeutic agents for chronic myelogenous leukemia.
- the present inventors have found that the CD147 antibody of the present invention activates p38 MAPK and SMAD signal transduction system to cancer cells.
- the present inventors have found that the CD147 antibody of the present invention shows excellent antitumor effect in SMAD4 positive cells.
- the present invention includes the following inventions.
- Human CD147 antibody or antigen binding fragment of said antibody (A) An antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 71, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 69, (B) an antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 51, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 49, (C) an antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 61, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 59, (D) an antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in
- Human CD147 characterized in that it binds to an epitope to which at least one antibody selected from the group consisting of the following (A) to (F) binds and which activates CD147-mediated signal transduction:
- Antibody or antigen binding fragment of said antibody (A) An antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 71, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 69, (B) an antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 51, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 49, (C) an antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 61, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 59, (D) an antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:
- [6] The antibody according to any one of [1] to [5] or the antigen of the antibody, which binds to an epitope comprising the residue from arginine 106 (Arg) to amino acid 165 (SEQ ID NO: 3) of SEQ ID NO: 3 Binding fragment.
- the heavy chain sequence comprises a variable region having CDRH1, CDRH2 and CDRH3, said CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 75, said CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 76, said CDRH3 Consisting of the amino acid sequence shown in SEQ ID NO: 77; and the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, said CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 72, said CDRL2 shown in SEQ ID NO: 73 Said amino acid sequence, wherein said CDRL3 consists of the amino acid sequence as shown in SEQ ID NO: 74;
- the antibody according to any one of [1] to [7] or an antigen-binding fragment of the antibody, which is characterized by [9] Any one of [1] to [5] which binds to an epitope comprising the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence in which one or several
- the heavy chain sequence comprises a variable region comprising CDRH1, CDRH2 and CDRH3, said CDRH1 consisting of the amino acid sequence as shown in SEQ ID NO: 55, said CDRH2 consisting of the amino acid sequence as shown in SEQ ID NO: 56, said CDRH3 being Consisting of the amino acid sequence as shown in No.
- the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, said CDRL1 consisting of the amino acid sequence as shown in SEQ ID NO: 52, said CDRL2 as shown in SEQ ID NO: 53 Said amino acid sequence, and said CDR L3 consists of the amino acid sequence shown in SEQ ID NO: 54;
- the antibody according to any one of [1] to [5] or 9, or an antigen-binding fragment of the antibody, which is characterized by
- the heavy chain sequence comprises a variable region comprising CDRH1, CDRH2 and CDRH3, said CDRH1 consisting of the amino acid sequence as shown in SEQ ID NO: 65, said CDRH2 consisting of the amino acid sequence as shown in SEQ ID NO: 66, said CDRH3 being Consisting of the amino acid sequence as shown in No.
- the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, said CDRL1 consisting of the amino acid sequence as shown in SEQ ID NO: 62, said CDRL2 as shown in SEQ ID NO: 63 Said amino acid sequence, said CDR L3 consisting of the amino acid sequence as shown in SEQ ID NO: 64;
- the antibody according to any one of [1] to [5] or 9, or an antigen-binding fragment of the antibody, which is characterized by [12]
- the heavy chain sequence comprises a variable region comprising CDRH1, CDRH2 and CDRH3, said CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 85, said CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 86, said CDRH3 Consisting of the amino acid sequence as shown in No.
- the light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, said CDRL1 consisting of the amino acid sequence as shown in SEQ ID NO: 82, said CDRL2 as shown in SEQ ID NO: 83 Said amino acid sequence, wherein said CDRL3 consists of the amino acid sequence as shown in SEQ ID NO: 84;
- the antibody of any one of [1] to [12], which is a chimeric antibody, is an antigen-binding fragment of the antibody.
- the heavy chain contains the constant region of the rabbit immunogullin B1 G1 heavy chain, the rabbit immunogum Brin G2 heavy chain, or the rabbit immune gout Gurine G4 heavy chain, and the light chain is the rabbit immunogut purine kappa light chain.
- a human CD147 antibody or an antigen-binding fragment of the antibody which comprises the following (c) and (d) and is characterized by activating CD147-mediated signal transduction:
- a heavy chain variable region consisting of the 20th to 136th amino acid residues of the amino acid sequence shown in SEQ ID NO: 135, and a light chain variable region consisting of the 21st to 128th amino acid residues of the amino acid sequence shown in SEQ ID NO: 137
- a heavy chain variable region consisting of the 20th to 136th amino acid residues of the amino acid sequence shown in SEQ ID NO: 147, and a light chain variable region consisting of the 21st to 128th amino acid residues of the amino acid sequence shown in SEQ ID NO: 149 The antibody according to [23] or an antigen-binding fragment of the antibody, which comprises [27] A heavy chain consisting of the 20th to 463th amino acid residues of the amino acid sequence shown in SEQ ID NO: 147 and a light chain consisting of the 21st to 234th amino acid residues of the amino acid sequence shown in SEQ ID NO: 149 [23 Or an antigen-binding fragment of the antibody.
- a heavy chain variable region consisting of the 20th to 136th amino acid residues of the amino acid sequence shown in SEQ ID NO: 147, and a light chain variable region consisting of the 21st to 128th amino acid residues of the amino acid sequence shown in SEQ ID NO: 151 The antibody according to [23] or an antigen-binding fragment of the antibody, which comprises [29] A heavy chain consisting of amino acid residues 20 to 463 of the amino acid sequence shown in SEQ ID NO: 147, and a light chain consisting of amino acid residues 21 to 234 in the amino acid sequence shown in SEQ ID NO: 151 [23 Or an antigen-binding fragment of the antibody.
- a human CD147 antibody or an antigen-binding fragment of the antibody which comprises the following (a) and (b) and is characterized by activating CD147-mediated signal transduction:
- the antibody or antigen-binding fragment of the antibody which comprises a light chain variable region consisting of amino acid residues 21 to 128 of the amino acid sequence shown in SEQ ID NO: 127.
- a human CD147 antibody or an antigen-binding fragment of the antibody which comprises the following (e) and (f) and is characterized by activating signaling via CD147:
- the antibody or antigen-binding fragment of the antibody which comprises a light chain consisting of amino acid residues 21 to 234 of the amino acid sequence shown.
- a human CD147 antibody or an antigen-binding fragment of the antibody which comprises the following (g) and (h) and is characterized by activating signaling via CD147:
- a heavy chain variable region consisting of the 20th to 138th amino acid residues of the amino acid sequence shown in SEQ ID NO: 139 and a light chain variable region consisting of the 21st to 128th amino acid residues of the amino acid sequence shown in SEQ ID NO: 141 The antibody according to [36] or an antigen-binding fragment of the antibody, which comprises [38] Comprising a heavy chain consisting of the 20th to 464th amino acid residue of the amino acid sequence shown in SEQ ID NO: 139 and a light chain consisting of the 21st to 234th amino acid residues of the amino acid sequence shown in SEQ ID NO: 141 [36 Or an antigen-binding fragment of the antibody.
- a pharmaceutical composition comprising at least any one of the antibody according to any one of [1] to [41] or an antigen-binding fragment of the antibody.
- the pharmaceutical composition according to [42] which is an antitumor agent.
- the pharmaceutical composition according to [43], wherein the tumor is a tumor that expresses CD147.
- Tumors are pancreatic cancer, liver cancer, gastric cancer, colon cancer, renal cancer, breast cancer, uterine cancer, ovarian cancer, lung cancer, lymphoma, thyroid cancer, skin cancer, head and neck cancer, sarcoma, prostate cancer, bladder cancer, brain tumor, digestive tract Stromal tumor (GIST), leukemia, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), malignant lymphoma, B cell lymphoma, non-Hodgkin
- AML acute myeloid leukemia
- CML chronic myelogenous leukemia
- CLL chronic lymphocytic leukemia
- ALL acute lymphocytic leukemia
- malignant lymphoma B cell lymphoma
- non-Hodgkin The pharmaceutical composition according to [43] or [44], which is lymphoma or diffuse large B-cell lymphoma (DLBCL
- Tumors include pancreatic cancer, liver cancer, gastric cancer, colon cancer, renal cancer, leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), The pharmaceutical composition according to any one of [43] to [45], which is malignant lymphoma, B cell lymphoma, non-Hodgkin's lymphoma or diffuse large B cell lymphoma (DLBCL).
- AML acute myelogenous leukemia
- CML chronic myelogenous leukemia
- CLL chronic lymphocytic leukemia
- ALL acute lymphocytic leukemia
- the pharmaceutical composition according to any one of [43] to [45] which is malignant lymphoma, B cell lymphoma, non-Hodgkin's lymphoma or diffuse large B cell lymphoma (DLBCL).
- Tumors are pancreatic cancer, liver cancer, gastric cancer, colon cancer, renal cancer, breast cancer, uterine cancer, ovarian cancer, lung cancer, lymphoma, thyroid cancer, skin cancer, head and neck cancer, sarcoma, prostate cancer, bladder cancer, brain tumor, digestive tract Stromal tumor (GIST), leukemia, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), malignant lymphoma, B cell lymphoma, non-Hodgkin
- AML acute myeloid leukemia
- CML chronic myelogenous leukemia
- CLL chronic lymphocytic leukemia
- ALL acute lymphocytic leukemia
- malignant lymphoma B cell lymphoma
- non-Hodgkin non-Hodgkin
- Tumors include pancreatic cancer, liver cancer, gastric cancer, colon cancer, renal cancer, leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL),
- AML acute myelogenous leukemia
- CML chronic myelogenous leukemia
- CLL chronic lymphocytic leukemia
- ALL acute lymphocytic leukemia
- the therapeutic method according to [49] to [51] which is malignant lymphoma, B cell lymphoma, non-Hodgkin's lymphoma or diffuse large B cell lymphoma (DLBCL).
- [54] The treatment method according to any one of [49] to [53], which is administered in combination with another antitumor agent.
- [55] A polynucleotide encoding the antibody according to any one of [1] to [41] or a functional fragment of the antibody.
- [61] A host cell transformed by the expression vector described in [60].
- p38 MAPK activation and / or SMAD4 activation increase p38 MAPK expression level, p38 MAPK phosphorylation level, HSP27 phosphorylation level, CXCL8 expression level increase, rhoB expression level increase, KLF5 mRNA reduction or KLF5 protein expression
- the antibody or a functional fragment of the antibody which is a reduced amount.
- a method for treating a tumor comprising administering the antibody according to [64] or an antigen-binding fragment of the antibody.
- a patient having a detected SMAD4 expression or KLF5 expression contained in the biological sample using a biological sample derived from a cancer patient and having detected SMAD4 or a detected decreased or deleted KLF5 expression To the treatment of cancer with the antibody according to any one of [1] to [41] or a functional fragment of the antibody or the pharmaceutical composition according to any one of [42] to [48].
- a method of predicting responsiveness to treatment of cancer comprising determining that it is responsive.
- a bispecific antibody comprising an antigen-binding fragment of the antibody according to any one of [1] to [41] and an antigen-binding fragment that binds to an antigen other than CD147.
- the antibody of the present invention is an antibody that specifically recognizes CD147, and is characterized by activating a related signal molecule mediated by CD147 and having high antitumor activity.
- CD147 is expressed not only on tumor cells but also on blood cells, the antibody of the present invention does not act on T cells and PBMCs and does not depend on effector function, so in development as an antitumor agent, It has the advantage of less safety concerns.
- the antibody of the present invention exhibits extremely high antitumor activity.
- the antibody of the present invention exhibits significantly higher efficacy in liver cancer cells than sorafenib, which is used as one of the standard therapeutic agents for liver cancer.
- the antibodies of the present invention exhibit significantly greater efficacy in pancreatic cancer cells than gemcitabine, which is used as one of the standard treatments for pancreatic cancer.
- the antibody of the present invention exhibits significantly higher efficacy in chronic myelogenous leukemia cells than imatinib, which is used as one of the standard therapeutic agents for chronic myelogenous leukemia.
- variable regions of (a) humanized antibody heavy chain # 131H2h and (b) humanized antibody light chain # 131L2h.
- Amino acid sequence and nucleotide sequence of # 110 L4 h Amino acid sequence and nucleotide sequence of # 110 L2h. Amino acid sequence and nucleotide sequence of # 110 L12 h. Amino acid sequence and nucleotide sequence of # 131H2h IgG2. Amino acid sequence and nucleotide sequence of # 131L2h.
- CD147 is black and antibody heavy chain (H CHAIN) and light chain (L CHAIN) are gray. Interaction surface of CD147 with antibody.
- the amino acids of CD147 near the antibody are represented by a stick model and are labeled with letters. Other parts of CD147 are represented by the black ribbon model.
- the amino acids of the antibody in the vicinity of CD147 are represented by a thin line model, and the other sites are represented by a gray ribbon model.
- CML chronic myelogenous leukemia
- Antitumor effect of humanized CD147 antibody in kidney cancer model Antitumor effect of humanized CD147 antibody in acute myeloid leukemia (AML) model. Antitumor effect of humanized CD147 antibody in pancreatic cancer model. Results of competition ELISA. Comparison of the antitumor effect of humanized CD147 antibody using (a) MIA PaCa-2 cells and (b) MIA PaCa-2 cells expressing KLF5.
- cancer and “tumor” are used in the same meaning, unless otherwise specifically limited, when solid cancer, non-solid cancer or both of them are intended.
- the term "gene” includes not only DNA but also its mRNA, cDNA and its cRNA.
- polynucleotide or “nucleotide” is used interchangeably with nucleic acid and also includes DNA, RNA, probes, oligonucleotides, and primers.
- polypeptide and “protein” are used interchangeably.
- the "cell” also includes cells in an animal individual and cultured cells.
- CD147 is used interchangeably with CD147 protein.
- “functional fragment of antibody” is also referred to as "antigen-binding fragment of antibody” and means a partial fragment of antibody having an antigen-binding activity, Fab, F (ab ') 2, Fv, scFv, diabody, linear antibodies, multispecific antibodies formed from antibody fragments, etc.
- Fab ' which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the antigen-binding fragment of the antibody.
- antigen binding fragments include not only those obtained by treating the full-length molecule of antibody protein with an appropriate enzyme, but also proteins produced in an appropriate host cell using a genetically engineered antibody gene.
- effector activity refers to antibody-dependent-cellular cytotoxicity (hereinafter referred to as ADCC) activity, complement-dependent cytotoxicity (hereinafter referred to as CDC) activity, Alternatively, it refers to any one or more of antibody-dependent cellular phagocytosis (hereinafter, referred to as ADCP) activity.
- ADCC antibody-dependent-cellular cytotoxicity
- CDC complement-dependent cytotoxicity
- ADCP antibody-dependent cellular phagocytosis
- effector function means that each "effector activity” is exerted.
- ADCC activity is characterized by immune cells with effector activity, antibodies and 51 Cr. It can be measured by the method of 51 Cr release assay that measures cell death that occurs when the labeled target cells are brought into contact.
- the ADCC activity of the human CD147 antibody of the present invention is measured as follows. For ADCC activity of human CD147 antibody to be evaluated, human peripheral blood mononuclear cells (PBMC) as effector cells, and CD147-positive human cancer cell line (eg, pancreatic cancer strain MIA PaCa-2) as ADCC target cells are used. In radioisotope 51 Cr and labeled cancer cell antibodies evaluated 0.5 or, 4 ° C.
- % Specific release (Total release-Spontaneous release) / Maximamu release Complement dependent cytotoxicity (CDC) activity can be evaluated by measuring cell death that occurs when contacting complement contained in blood, antibody and target cells.
- the CDC activity of the human CD147 antibody of the present invention is measured as follows.
- the complement dependent cell killing activity (CDC activity) by the human CD147 antibody to be evaluated is evaluated using human pancreatic strain MIA PaCa-2 as a target cell.
- a commercially available rabbit complement (Low Tox-M Rabbit Complement, CEDARLANE LABORATORIES LIMITED, Cat. CL3051) is used as a complement.
- Human IgG hIgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat.
- 009-000-003 is used as a CDC activity negative control antibody.
- the final concentration of rabbit complement will be 7.5%.
- intracellular ATP contained in living cells was measured using CellTiter-Glo Lumimescent Cell Viability Assay (Promega, Cat. G7572).
- the luminescent signal obtained using the CellTiter-Glo Lumimescent Cell Viability Assay is quantified using EnVision 2104 Multilabel Reader (Perkin Elmer). The measurements are performed in triplicate and the mean and standard deviation are calculated.
- the luminescence signal obtained from untreated cells is taken as 100%, and the luminescence signal reduced in a complement dependent manner as the antibody and CDC activity.
- Antibody-dependent cellular phagocytosis (ADCP) activity can be measured by dual fluorescence labeling of phagocytosis caused by contacting a phagocytotic immune cell, an antibody and a target cell.
- ADCP antibody-dependent cellular phagocytosis
- the ADCP activity of the human CD147 antibody of the present invention is measured as follows. Human IgG antibodies have been reported to exhibit cell killing activity against cancer cells by inducing antibody-dependent phagocytosis (ADCP) by monocytes and macrophages through interaction with mouse Fc ⁇ receptor (Overdijk et al., Journal of Immunology, 1-9, 2012).
- the ADCP activity of the human chimeric antibody of the present invention is evaluated using RAW 264.7 (ATCC, TIB-71) as an effector cell and human pancreatic strains PANC-1 or MIA PaCa-2 as an ADCP target cell.
- a flow cytometer (BD, Canto II) the percentage of PKH26 positive cells that have transferred to PKH67 signal positive by phagocytosis is measured.
- the measurement is similarly carried out on a sample treated with human IgG (hIgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) as a negative control sample.
- the measurement is performed in triplicate, and the average value and standard deviation are calculated.
- “having substantially no effector activity” or “effector activity reduced or deleted” means that the antibody has at least one of ADCC activity, CDC activity or ADCP activity. Or have a low degree of activity such that their function can not be fully exerted.
- the phrase "having substantially no effector activity” or “having or without effector activity” means, for example, that the activity of the antibody to be assessed is as active as the negative control in the above-mentioned activity assessment method.
- the phrase "the ADCC activity has been reduced or deleted” means that the antibodies to be evaluated do not exhibit ADCC activity, or have such a low level of activity that their functions can not be sufficiently exhibited.
- ADC activity is reduced or deleted means, for example, that the activity of the antibody to be evaluated is as active as the negative control in the above-mentioned activity evaluation method.
- CDC activity is reduced or deleted means that the antibodies to be evaluated do not exhibit CDC activity or have a low level of activity such that their functions can not be sufficiently exhibited.
- the phrase “CDC activity is reduced or deleted” means, for example, that the activity of the antibody to be evaluated is as active as the negative control in the above-mentioned activity evaluation method.
- ADCP activity has been reduced or deleted means that the antibodies to be evaluated do not exhibit ADCP activity or have such a low level of activity that their functions can not be sufficiently exhibited.
- ADCP activity is reduced or deleted means, for example, that the activity of the antibody to be evaluated is as active as the negative control in the above-mentioned activity evaluation method.
- DCC activity reduced or deleted refers, for example, to the antibodies to be evaluated in the aforementioned activity evaluation methods, respectively.
- the activity is the same as that of the negative control.
- activate signal transduction via CD147 means via CD147.
- Activation of CD147-mediated signal transduction means that expression of a gene downstream of CD147 signal is enhanced or reduced, protein expression is enhanced or reduced, or protein phosphorylation is enhanced or reduced.
- Related signal molecules downstream of CD147 include, for example, activation of FAK, MEK, Erk, JAK / STAT, AKT or MAP kinase (MAPK), or signal molecules further downstream thereof.
- MAPKs include, for example, ERK1 / 2, JNK or p38 MAPK, and more preferably p38 MAPK.
- Signal molecules further downstream of MAPK include, for example, HSP27, cxcl8 or SMAD (eg, SMAD2, SMAD3 and / or SMAD4).
- activation of CD147 for example, increase of mRNA expression level of p38 MAPK, increase of protein expression level of p38 MAPK, phosphorylation of p38 MAPK, phosphorylation of HSP27 (for example, phosphorylation of Ser27 of HSP27 or Ser15 of HSP27) Phosphorylation), increase of cxcl8 mRNA expression level, increase of cxcl8 protein expression level, increase of rhoB mRNA expression level through SMAD signal activation or increase of rhoB protein expression level, or reduction of KLF5 mRNA or KLF5 protein expression level Decrease in
- epitope means a partial peptide or partial conformation of CD147 to which a specific anti-CD147 antibody binds.
- the epitope which is a partial peptide of the above-mentioned CD147 can be determined by a method well known to those skilled in the art such as an immunoassay.
- various partial structures of the antigen are prepared.
- known oligonucleotide synthesis techniques can be used. For example, after producing a series of polypeptides which are sequentially shortened by an appropriate length from the C terminus or N terminus of CD147 using gene recombination technology well known to those skilled in the art, the reactivity of the antibody against them is examined.
- epitopes can be determined by synthesizing further shorter peptides and examining their reactivity with the peptide.
- an antibody that binds to a membrane protein consisting of a plurality of extracellular domains has, as an epitope, a three-dimensional structure consisting of a plurality of domains, the three-dimensional structure is altered by modifying the amino acid sequence of a specific extracellular domain. It is possible to determine which domain to bind to.
- An epitope which is a partial conformation of an antigen to which a specific antibody binds can also be determined by identifying amino acid residues of the antigen adjacent to the above antibody by X-ray structural analysis.
- the second antibody binds to the partial peptide or partial conformation to which the first antibody binds, it can be determined that the first antibody and the second antibody have a common epitope. Also, by confirming that the second antibody cross competes for binding of the first antibody to the antigen (ie, the second antibody interferes with the binding of the first antibody to the antigen), the specific epitope Even if the sequence or structure is not determined, it can be determined to bind to the same epitope of the first antibody and the second antibody. When the first antibody and the second antibody bind to the same epitope, and the first antibody has a special effect such as anti-tumor activity, it can be expected that the second antibody also has similar activity.
- CDRs complementarity determining regions
- the complementarity-determining region also called hypervariable region, is a site within the variable regions of the antibody heavy and light chains, where the variability of the primary structure is particularly high, and the heavy and light chains are The primary chain of the polypeptide chain is separated into three parts each.
- CDRH1, CDRH2, CDRH3 from the amino terminal side of the heavy chain amino acid sequence
- the complementarity determining region of the light chain is The amino acid side of the chain amino acid sequence.
- “several” when there are descriptions “one to several” and “one or several” indicates two to ten. Preferably, it is 10 or less, more preferably 5 or 6 or less, and even more preferably 2 or 3.
- CD147 is a single-pass transmembrane protein with 2 to 3 immunoglobulin-like domains and interacts with CD147, such as CD44, Integrin family molecules, CD98, VEGFR, CypA / B, MCT1 / 3/4 proliferation, invasion, By interacting with molecules on the extracellular or cell membrane surface involved in inflammation, downstream signal related molecules, FAK, MEK, Erk, JAK / STAT, AKT, MAPK family molecules are activated, and MMP and other proteases are produced It is known to promote cancer growth, metastasis, and invasion.
- Variant 1 is a single-pass transmembrane protein with three immunoglobulin-like domains (each referred to herein as D0, D1 and D2, respectively) expressed specifically in the retina
- variant 2 is T 1-transmembrane protein with two immunoglobulin-like domains (D1 and D2) that are expressed in cells and various normal cells and reported to have increased expression in various cancer tissues; It is a one time transmembrane protein with two immunoglobulin-like domains.
- the amino acid sequence and nucleotide sequence of variant 1 of human CD147 can be obtained by referring to GenBank Accession Nos. NP_001719.2, NM_001728.3, wherein the amino acid sequence is SEQ ID NO: 1 and the nucleotide sequence is the sequence It is also disclosed as number 2.
- the three immunoglobulin-like domains of variant 1 are amino acid numbers 22 to 138 (D0), amino acid numbers 140 to 218 (D1) and amino acid numbers 223 to 323 (D2), respectively, according to SEQ ID NO: 1 (Redzic) J., J. Mol. Biol., 2011, 68-82) (Grass et al., Biosol. Rep, 2016, 1-16).
- the transmembrane domain of variant 1 is amino acid numbers 324 to 344 in SEQ ID NO: 1.
- the amino acid sequence and nucleotide sequence of variant 2 of human CD147 can be obtained by referring to GenBank Accession Nos. NP_940991.1, NM_198589.2, wherein the amino acid sequence is SEQ ID NO: 3 and the nucleotide sequence is the sequence It is also disclosed as number 4.
- the two immunoglobulin-like domains (D1, D2) of variant 2 are amino acid numbers 24 to 102 (D1) and amino acid numbers 107 to 207 (D2), respectively, according to SEQ ID NO: 3.
- the transmembrane domain of variant 2 is amino acid numbers 208 to 228 in terms of SEQ ID NO: 3 (Grass et al., Biosol. Rep, 2016, 1-16).
- the amino acid sequence and nucleotide sequence of variant 3 of human CD147 can be obtained by referring to GenBank Accession Nos. NP_940992.1, NM_198590.2.
- the human CD147 gene can also be obtained from commercial sources.
- the amino acid sequence and nucleotide sequence of cynomolgus monkey CD147 (which may be referred to as monkey CD147 herein) can be obtained by referring to GenBank accession numbers XP_005587354.1, XM_005587297.1.
- the monkey CD147 gene can also be obtained from commercial sources.
- the amino acid sequence and nucleotide sequence of mouse CD147 can be obtained by referring to GenBank Accession Nos. NP — 001070652.1, NM — 001077184.1.
- the mouse CD147 gene can also be obtained from commercial sources.
- CD147 used in the present invention can be obtained by in vitro synthesis or production in host cells by genetic engineering. Specifically, after integrating CD147 cDNA into an expressible vector, it is synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryotic or eukaryotic host cells The protein can be obtained by expressing CD147 by transforming E. coli.
- the cDNA of CD147 can be prepared, for example, by polymerase chain reaction (hereinafter referred to as "PCR") using a primer that specifically amplifies cDNA of CD147, using the cDNA library expressing cDNA of CD147 as a template and the like.
- PCR polymerase chain reaction
- K., et al., Science, (1988) 239, 487-49) can be obtained by the so-called PCR method.
- a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding human, monkey or mouse CD147, and which encodes a protein having a biological activity equivalent to that of CD147 Is also contained in the cDNA of CD147.
- a splicing variant transcribed from the human, monkey or mouse CD147 locus or a polynucleotide which hybridizes to the same under stringent conditions, and which encodes a protein having a biological activity equivalent to that of CD147 is also useful. It is contained in the cDNA of CD147.
- the amino acid sequence of human, monkey or mouse CD147 or an amino acid sequence from which a signal sequence has been removed, consists of an amino acid sequence in which one or several amino acids are substituted, deleted or added.
- a protein having the same biological activity as that of is also contained in CD147.
- an amino acid sequence encoded by a splicing variant transcribed from human, monkey or mouse CD147 locus or an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence, and And a protein having biological activity equivalent to that of CD147 are also included in CD147.
- the antibody to CD147 of the present invention immunizes a non-human animal with a target antigen, and the lymph, lymph tissue, blood cell sample or bone marrow-derived cells are collected from the immunized animal, and known methods (eg Kohler and Milstein, Antibody-producing cells producing antibodies against CD147 according to Nature (1975) 256, p. 495-497, Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N. Y. (1980)).
- a hybridoma can be established by fusing E. coli and myeloma cells to obtain a monoclonal antibody.
- Examples of the monoclonal antibody thus obtained include, for example, LN22R8, 2P10F2, rat_CD147_ # 84, rat_CD147_ # 101, rat_CD147_ # 110 or rat_CD147_ # 131.
- the method for obtaining a monoclonal antibody falls in the field already established, and is not limited to the above specific example.
- the antibody of the present invention may be a genetically engineered antibody, for example, a chimeric (chimeric) antibody, a humanized (for example, a chimeric (chimeric)) antibody, in addition to the above-mentioned monoclonal antibody against CD147, for the purpose of reducing heterologous antigenicity to human etc.
- Humanized antibodies, human antibodies and the like are also included. These antibodies can be produced using known methods.
- chimeric antibodies include antibodies in which the variable region of the antibody and the constant region are heterologous to each other, such as a chimeric antibody in which the variable region of a mouse- or rat-derived antibody is conjugated to a constant region derived from human (Proc. Natl. Acad) Sci.U.S.A., 81, 6851-6855, (1984)).
- a heavy chain having an amino acid sequence consisting of the 20th to 463rd amino acid residues of SEQ ID NO: 104 in the sequence listing, a 20th to 464th amino acid residue of SEQ ID NO: 106 in the sequence listing Or an amino acid sequence consisting of the 20th to 464th amino acid residues of SEQ ID NO: 108 and an amino acid sequence consisting of the 21st to 234th amino acid residues of SEQ ID NO: 102
- the antibody which consists of light chains is mentioned.
- the antibody which consists of light chains is mentioned.
- Examples include an antibody consisting of a heavy chain having a sequence and a light chain having an amino acid sequence consisting of the 21st to 234th amino acid residues of SEQ ID NO: 118.
- humanized antibody an antibody in which only a CDR is incorporated into a human-derived antibody (see Nature (1986) 321, p. 522-525), and by CDR grafting, amino acid residues of some frameworks in addition to the CDR sequences.
- the group may also be an antibody (International Publication WO 90/07861) grafted to a human antibody.
- the antibody of the present invention as long as it retains all six CDR sequences of rat_CD147_ # 84, has binding activity to CD147, and activates CD147.
- the heavy chain variable region of the rat_CD147_ # 84 antibody comprises CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 55, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 56, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 57 It holds.
- the light chain variable region of the rat_CD147_ # 84 antibody comprises CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 52, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 53, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 54 It holds.
- the amino acid and nucleotide sequences of the light chain variable region or heavy chain variable region of the rat_CD147_ # 84 antibody, and the amino acid sequences of the CDRs are also described in FIGS. 33-1 and 33-2.
- the antibody according to the present invention as long as it retains all six CDR sequences of rat_CD147_ # 101, has binding activity to CD147, and activates CD147.
- the heavy chain variable region of the rat_CD147_ # 101 antibody comprises CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 65, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 66, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 67 It holds.
- the light chain variable region of the rat_CD147_ # 101 antibody comprises CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 62, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 63, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 64 It holds.
- the amino acid and nucleotide sequences of the light chain variable region or heavy chain variable region of the rat_CD147_ # 101 antibody, and the amino acid sequences of the CDRs are also described in FIGS. 34-1 and 34-2.
- the antibody of the present invention as long as it retains all six CDR sequences of rat_CD147_ # 110, has binding activity to CD147, and activates CD147.
- the heavy chain variable region of the rat_CD147_ # 110 antibody comprises CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 75, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 76, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 77 It holds.
- the light chain variable region of the rat_CD147_ # 110 antibody comprises CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 72, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 73, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 74 It holds.
- the amino acid and nucleotide sequences of the light chain variable region or heavy chain variable region of the rat_CD147_ # 110 antibody, and the amino acid sequences of the CDRs are also described in FIGS. 35-1 and 35-2.
- the antibody of the present invention as long as it retains all six CDR sequences of rat_CD147_ # 131, has binding activity to CD147, and activates CD147 include.
- the heavy chain variable region of the rat_CD147_ # 131 antibody comprises CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 85, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 86, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 87 It holds.
- the light chain variable region of the rat_CD147_ # 131 antibody comprises CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 82, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 83, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 84 It holds.
- the amino acid and nucleotide sequences of the light or heavy chain variable region of the rat_CD147_ # 131 antibody, and the amino acid sequences of the CDRs are also described in FIGS. 36-1 and 36-2.
- CDR-modified humanized antibody in which 1 to 3 amino acid residues in each CDR are substituted with other amino acid residues is also an antibody that has binding activity to CD147 and activates CD147.
- CD147 a CDR-modified humanized antibody in which 1 to 3 amino acid residues in each CDR are substituted with other amino acid residues is also an antibody that has binding activity to CD147 and activates CD147.
- the humanized antibody derived from rat_CD147_ # 84 antibody includes human CD147 antibody or an antigen-binding fragment of the antibody, having the following (a) and (b): (A) The heavy chain variable region according to any one selected from the group consisting of (a1) to (a4) below: (A1) a heavy chain variable region consisting of the 20th to 140th amino acid residues of the amino acid sequence shown in SEQ ID NO: 123; (A2) a heavy chain variable region consisting of the 20th to 140th amino acid residues of the amino acid sequence shown in SEQ ID NO: 125; (A3) an amino acid sequence having at least 95% or more sequence identity to a sequence of a framework region other than each CDR sequence in the sequence of (a1) or (a2); and (a4) (a1) to (a3) An amino acid sequence in which one or several amino acids have been deleted, substituted or added in the sequence of a framework region other than each CDR sequence in any one of the sequences of (B) The light chain variable region according to any one
- a heavy chain variable region consisting of the 20th to 140th amino acid residues of the amino acid sequence shown in SEQ ID NO: 125 and 21 to 14 of the amino acid sequence shown in SEQ ID NO: 127 An antibody comprising a light chain variable region consisting of the 128th amino acid residue, or a heavy chain variable region consisting of the 20th to 140th amino acid residues of the amino acid sequence shown in SEQ ID NO: 123 and an amino acid sequence shown in SEQ ID NO: 127
- a heavy chain consisting of the 20th to 467th amino acid residues of the amino acid sequence shown in SEQ ID NO: 125 and 21 to 234 of the amino acid sequence shown in SEQ ID NO: 127 Antibody comprising a light chain consisting of the amino acid residue of SEQ ID NO: 1, or a heavy chain consisting of the amino acid residues at positions 20 to 466 of the amino acid sequence shown in SEQ ID NO: 123 and amino acids 21 to 234 in the amino acid sequence shown in SEQ ID NO: 127
- Humanized antibodies derived from rat_CD147_ # 101 antibody include human CD147 antibody or an antigen-binding fragment of the antibody, having the following (e) and (f):
- Preferred examples of the humanized antibody derived from rat_CD147_ # 101 antibody include a heavy chain variable region consisting of the 20th to 137th amino acid residues of the amino acid sequence shown in SEQ ID NO: 129 and 21 to 14 of the amino acid sequence shown in SEQ ID NO: 133
- An antibody comprising a light chain variable region consisting of the 128th amino acid residue, or a heavy chain variable region consisting of the 20th to 137th amino acid residues of the amino acid sequence shown in SEQ ID NO: 131 and an amino acid sequence shown in SEQ ID NO: 133
- Antibody comprising a light chain consisting of the amino acid residue of SEQ ID NO: 1, or a heavy chain consisting of the amino acid residues at positions 20 to 464 of the amino acid sequence shown in SEQ ID NO: 131 and amino acids 21 to 234 in the amino acid sequence shown in SEQ ID NO: 133
- antibodies comprising a light chain consisting of amino acid residues of
- the humanized antibody derived from rat_CD147_ # 110 antibody includes human CD147 antibody or an antigen-binding fragment of the antibody, which has the following (c) and (d): (C) The heavy chain variable region according to any one selected from the group consisting of (c1) to (c4) below: (C1) a heavy chain variable region consisting of the 20th to 136th amino acid residues of the amino acid sequence shown in SEQ ID NO: 135; (C2) a heavy chain variable region consisting of the 20th to 136th amino acid residues of the amino acid sequence shown in SEQ ID NO: 147; (C3) an amino acid sequence having at least 95% or more sequence identity to a sequence of a framework region other than each CDR sequence in the sequence of (c1) or (c2); and (c4) (c1) to (c3) An amino acid sequence in which one or several amino acids have been deleted, substituted or added in the sequence of a framework region other than each CDR sequence in any one of the sequences of (D) The light chain variable region according to
- a heavy chain variable region consisting of the 20th to 136th amino acid residues of the amino acid sequence shown in SEQ ID NO: 135 and 21 to 14 of the amino acid sequence shown in SEQ ID NO: 137 An antibody comprising a light chain variable region consisting of the 128th amino acid residue; a heavy chain variable region consisting of the 20th to 136th amino acid residue of the amino acid sequence shown in SEQ ID NO: 147 and 21 of the amino acid sequence shown in SEQ ID NO: 149
- An antibody comprising a light chain variable region consisting of amino acid residues 128 to 128; or a heavy chain variable region consisting of amino acid residues 20 to 136 of the amino acid sequence shown in SEQ ID NO: 147 and an amino acid shown in SEQ ID NO: 151 An antibody comprising a light chain variable region consisting of amino acid residues 21 to 128 of the sequence; .
- Antibody comprising a light chain consisting of the amino acid residue of SEQ ID NO: 127; heavy chain consisting of the amino acid residue consisting of amino acid residues 20 to 463 of the amino acid sequence shown in SEQ ID NO: 147; and 21 to 234 in the amino acid sequence shown in SEQ ID NO: 149
- antibodies comprising a light chain consisting of the second amino acid residue.
- the humanized antibody derived from rat_CD147_ # 131 antibody includes human CD147 antibody or an antigen-binding fragment of the antibody, which has the following (g) and (h): (G) The heavy chain variable region according to any one selected from the group consisting of (g1) to (g3) below: (G1) a heavy chain variable region consisting of the 20th to 138th amino acid residues of the amino acid sequence shown in SEQ ID NO: 139; (G2) an amino acid sequence having at least 95% or more sequence identity to a sequence of a framework region other than each CDR sequence in the sequence of (g1); and (g3) each in the sequence of (g1) or (g2) An amino acid sequence in which one or several amino acids have been deleted, substituted or added in the sequence of framework regions other than CDR sequences, and (H) The light chain variable region according to any one selected from the group consisting of (h1) to (h3) below: (H1) a light chain variable region consisting of the 21st to 128th amino acid residues of the
- a heavy chain variable region consisting of the 20th to 138th amino acid residues of the amino acid sequence shown in SEQ ID NO: 139 and 21 to 14 of the amino acid sequence shown in SEQ ID NO: 141 An antibody comprising a light chain variable region consisting of the 128th amino acid residue is mentioned.
- antibodies comprising a light chain consisting of the second amino acid residue As a more preferable example of the humanized antibody derived from rat_CD147_ # 131 antibody, a heavy chain consisting of the 20th to 464th amino acid residues of the amino acid sequence shown in SEQ ID NO: 139 and 21 to 234 of the amino acid sequence shown in SEQ ID NO: 141 And antibodies comprising a light chain consisting of the second amino acid residue.
- humanized antibody derived from rat_CD147_ # 84 antibody is preferably CD147-mediated Activate p38 MAPK signaling and / or SMAD4 signaling.
- the antibodies of the present invention can further include human antibodies.
- the anti-CD147 human antibody means a human antibody having only the gene sequence of the antibody derived from human chromosome.
- the anti-CD147 human antibody can be prepared, for example, by using a human antibody-producing mouse having a human chromosomal fragment containing heavy and light chain genes of human antibody (Tomizuka, K. et al., Nature Genetics (1997) 16, p. Kuroda, Y. et. Al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et. Al., Animal Cell Technology: Basic and Applied Aspects vol. , P.
- HAC human artificial chromosome
- Mouse artificial mouse artificial chromosome
- eukaryotic cells are transformed with a cDNA encoding each of heavy and light chains of such human antibody, preferably a vector containing the cDNA, by genetic recombination technology to produce a recombinant human monoclonal antibody.
- This antibody can also be obtained from the culture supernatant by culturing transformed cells.
- eukaryotic cells preferably CHO cells
- mammalian cells such as lymphocytes and myelomas can be used as lymphocytes and myelomas.
- a method for obtaining a phage display-derived human antibody selected from a human antibody library (Wormstone, IM et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), p. 2301-2308; Mé, S. et. Al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; Siriwardena, D. et. Al., Ophthalmology (2002) 109 (3), p. 427-431, etc.) are also known.
- a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105) in which the variable region of human antibody is expressed on the phage surface as a single chain antibody (scFv) and phages that bind to the antigen are selected. -1116) can be used.
- the DNA sequence encoding the variable region of human antibody binding to the antigen can be determined.
- a human antibody can be obtained by preparing an expression vector having the sequence and introducing it into a suitable host for expression (WO 92/01047, WO 92 / 20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388, Annu. Rev. Immunol (1994) 12, p. 433-455, Nature Biotechnology (2005) 23 (9), p. 1105-1116).
- an antibody having the same epitope as the antibody provided by the present invention is also included in the antibody of the present invention.
- an antibody having the same epitope as at least any one of LN22R8, 2P10F2, rat_CD147_ # 84, rat_CD147_ # 101, rat_CD147_ # 110 or rat_CD147_ # 131 can be mentioned.
- the LN22R8 and 2P10F2 of the present invention recognized the epitope represented by mu3 in FIG. 3 (human CD147v1: DALPGQKTEFKVDDDDQ (SEQ ID NO: 143), monkey CD147: DTLPGQKTDFEVDSDDL (SEQ ID NO: 144)).
- human CD147v1 DALPGQKTEFKVDDDDQ (SEQ ID NO: 143)
- monkey CD147 DTLPGQKTDFEVDSDDL (SEQ ID NO: 144)
- one or several, preferably 1 to 3, more preferably 1 or 2 amino acids are deleted in the sequence of SEQ ID NO: 143 or SEQ ID NO: 144, or SEQ ID NO: 143 or SEQ ID NO: 144
- this antibody activates signaling via CD147.
- the antibodies of the present invention include antibodies that recognize the same epitope as rat_CD147_ # 110, preferably humanized # 110H1L4. Preferably, this antibody activates signaling via CD147.
- the results of epitope analysis of humanized # 110 H1 L4 are shown in Example 17.
- Suitable antibodies can be selected by evaluating their binding to antigens well known to those skilled in the art.
- the dissociation constant between the antibody and the antigen (CD147) can be measured using Biacore T200 (GE Healthcare Bioscience), whose principle is surface plasmon resonance (SPR).
- SPR surface plasmon resonance
- the evaluation of binding to CD147 is not limited to the use of Biacore T200, but is an instrument based on surface plasmon resonance (SPR) as a detection principle, KinExA (Sapidyne Instruments) based on a binding exclusion assay (Kinetic Exclusion Assay), It is also possible to use a BLitz system (Pall), which uses Bio-Layer Interferometry as a detection principle, or an ELISA (Enzyme-Linked Immunosorbent Assay) method or the like.
- SPR surface plasmon resonance
- KinExA Stemetic Exclusion Assay
- BLitz system Pall
- ELISA Enzyme-Linked Immunosorbent Assay
- DSC Differential scanning calorimetry
- Tm heat denaturation midpoint
- the storage stability of the antibody is known to show some correlation with the thermal stability of the antibody (Lori Burton, et. Al., Pharmaceutical Development and Technology (2007) 12, p. 265-273).
- Suitable antibodies can be selected on the basis of stability.
- Other indicators for selecting antibodies can include high yields in appropriate host cells and low aggregation in aqueous solution. For example, since the antibody with the highest yield does not necessarily exhibit the highest thermostability, it is necessary to select the antibody most suitable for human administration, judging comprehensively on the basis of the index described above. .
- single chain immunoglobulin by linking the full-length sequences of antibody heavy chain and light chain using an appropriate linker (Lee, HS, et. al., Molecular Immunology (1999) 36, p. 61-71; Shirrmann, T. et al., mAbs (2010), 2, (1) p. 1-4).
- linker Lee, HS, et. al., Molecular Immunology (1999) 36, p. 61-71; Shirrmann, T. et al., mAbs (2010), 2, (1) p. 1-4.
- Such single-chain immunoglobulins can retain their structure and activity similar to that of an antibody that is originally tetramer by dimerizing.
- the antibody of the present invention may be an antibody having a single heavy chain variable region and no light chain sequence.
- Such an antibody is called a single domain antibody (sdAb) or a nanobody, and is actually observed in camels or llamas, and it is reported that the antigen binding ability is maintained.
- sdAb single domain antibody
- the above antibody can also be interpreted as one of the antigen-binding fragments of the antibody in the present invention.
- WO 99/54342, WO 2000/61739, WO 2002/31140, etc. are known as techniques for modulating sugar chain modification of antibody, but are not limited thereto.
- a combination of a suitable host and an expression vector can be used.
- antibody genes As specific examples of antibody genes, mention may be made of a combination of a gene encoding the heavy chain sequence of the antibody described herein and a gene encoding the light chain sequence.
- the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or can be inserted into separate expression vectors. is there.
- eukaryotic cells animal cells, plant cells, and eukaryotic microorganisms can be used.
- animal cells mammalian cells, for example, cells of monkeys such as COS cells (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblast NIH 3 T 3 (ATCC No.
- CRL Dihydrofolate reductase deficient strain (Urlaub, G. and Chasin, L. A. Proc. Natl. Acad. Sci. U. S. A.) of Chinese hamster ovary cells (CHO cells, ATCC CCL-61). (1980) 77, p. 4126-4220). Moreover, when using a prokaryote, E. coli and Bacillus subtilis can be mentioned, for example. The antibody gene of interest is introduced into these cells by transformation, and the transformed cells are cultured in vitro to obtain an antibody. In the above culture methods, the yield may differ depending on the sequence of the antibody, and it is possible to select from among the antibodies having the same binding activity, those which can be easily produced as medicaments using the yield as an index.
- IgG IgG1, IgG2, IgG3, IgG4
- IgM IgA (IgA1, IgA2)
- IgD or IgE etc. can be mentioned, preferably IgG or IgM, More preferably, IgG is mentioned.
- human IgG1 has very strong effector functions such as CDC activity via complement binding and antibody-dependent cellular cytotoxicity (Bruggemann et al., J. Exp. Med , 1351-1361, 1987), when targeting molecules that are highly expressed in cancer with therapeutic antibodies, an IgG format that exhibits a therapeutic effect by promoting induction of cell death in cancer cells by effector function-mediated cytotoxicity. It is used as (trastuzumab, rituximab etc).
- IgG1 antibodies targeted to HLA-DR depend on the CDC activity of the antibodies contained in human IgG1, and it has been reported that cynomolgus monkeys died after administration, and targeting molecules expressed even in normal organs In antibody drugs, the effector function may cause serious side effects (Tawara, T., J. Immunology, 2008, 2294-2298).
- the IgG1 antibody may have a mutation, and it is possible to modulate effector function by substituting a part of amino acid residues in the constant region. (See WO 88/007089, WO 94/28027, WO 94/29351).
- IgG1 mutants having attenuated effector functions include IgG1 LALA (IgG1-L234A, L235A), 1gG1 LAGA (IgG1-L235A, G237A) and the like.
- human IgG2 has very weak effector functions such as CDC activity via complement binding and antibody-dependent cellular cytotoxicity (Bruggemann et al., J. Exp. Med. , 1351-1361, 1987), which is used as one of the IgG formats for avoiding toxicity due to cytotoxicity via effector function when targeting a molecule expressed in a normal organ with a therapeutic antibody (denosumab , Eborokumab, Brodalumab).
- human IgG 4 has very weak effector functions such as CDC activity via complement binding and antibody-dependent cellular cytotoxicity (Bruggemann et al., J. Exp. Med. , 1351-1361, 1987), which is used as one of the IgG formats for avoiding the toxicity due to the cytotoxicity caused by the effector function when targeting a molecule expressed in a normal organ with a therapeutic antibody (opsybo ).
- IgG4 is used as the isotype of the antibody of the present invention, partial substitution of amino acid residues in the constant region can suppress the resolution peculiar to IgG4 and prolong its half life (Molecular Immunology, 30, 1 105-108 (1993)).
- the IgG4 antibody may have a mutation.
- IgG4 it is position 234 shown by EU index (Proc Natl Acad Sci US A. 1969, 63 (1), 78-85; Kabat et. Al., Sequences of proteins of immunological interest, 1991 Fifth edition).
- EU index Proc Natl Acad Sci US A. 1969, 63 (1), 78-85; Kabat et. Al., Sequences of proteins of immunological interest, 1991 Fifth edition.
- F234A phenylalanine to alanine
- L235A leucine at position 235 to alanine
- Such antibody mutations are called FALA mutations.
- IgG4 PFALA further attenuates effector function by replacing two amino acid residues necessary for interaction with Fc ⁇ Rs (for example, Fc ⁇ RI, Fc ⁇ RII or Fc ⁇ RIII) present in the CH2 domain with alanine.
- Fc ⁇ Rs for example, Fc ⁇ RI, Fc ⁇ RII or Fc ⁇ RIII
- a mutation is introduced to promote the formation of SS bonds between antibody heavy chains in order to enhance stability.
- Such mutations include substitution of the serine at position 228 with a proline (S228P) indicated by the EU index (ANGAL et. Al., Molecular Immunology, 105-108, 1993). Mutation of this antibody is called Pro mutation.
- the above-mentioned FALA mutation and Pro mutation may be introduced simultaneously into the constant region of the antibody of the present invention (Vafa et. Al., Methods, 65, 114-126, 2014).
- An IgG4 heavy chain having a FALA mutation is referred to as an "IgG4 FALA” type heavy chain
- an IgG4 heavy chain having a Pro mutation is referred to as an "IgG4P” type heavy chain
- an IgG4 heavy chain having both a FALA mutation and a Pro mutation is referred to as "the Called "IgG 4 PFALA" type heavy chain.
- the antibody heavy chain constant region consists of CH1, hinge, CH2 and CH3 regions, where CH1 is EU index 118 to 215, hinge is EU index 216 to 230, CH2 is EU index 231 to 340, CH3 is EU index 341 to 446 and It is defined.
- SEQ ID NO: 100 the 248th proline, 254th alanine and 255th alanine, the amino acid sequence of human chimeric rat_CD147_ # 101 heavy chain IgG4 PFALA, in SEQ ID NO: 108, the 245th proline, 251st alanine And 252nd alanine, 244th proline in SEQ ID NO: 108 showing the amino acid sequence of human chimeric rat_CD147_ # 110 heavy chain IgG4 PFALA, 250th Corresponding to alanine and 251 th alanine.
- Preferred isotypes of the antibody of the present invention include IgG1, IgG2, IgG4, IgG4P or IgG4 PFALA, particularly preferably IgG2, IgG4P or IgG4 PFALA, and still more preferably IgG2 or IgG4P.
- the antibody of the present invention may also be an antigen-binding fragment of an antibody having an antigen-binding portion of the antibody or a modified product thereof.
- a fragment of the antibody can be obtained by treating the antibody with a proteolytic enzyme such as papain or pepsin, or modifying the antibody gene by genetic engineering techniques and expressing it in appropriate cultured cells.
- a fragment retaining all or part of the function of the antibody full-length molecule can be referred to as an antigen-binding fragment of an antibody.
- the function of the antibody can include activation of signaling associated with the antigen.
- CD147 is also expressed in blood cells including erythrocytes and normal organs essential for survival (Spring, et al., Eur. J. Immunol., 1997, 891-897), antibody-associated effector functions
- the anti-tumor effect of using is considered to be at high risk of side effects.
- erythrocytes are sensitive to effector functions (ADCC, CDC, ADCP) generated by antibody binding (Flegel, W., Transfusion, 2015, S47-S58), and antibodies against erythrocytes increase in the body Is known to result in autoimmune hemolytic anemia (Gibson, J., Aust. N. Z. J. Med., 1988. 625-637).
- the antibody of the present invention is a therapeutic antibody that targets CD147 that is also expressed in normal cells, and detects any or all of ADCC activity, ADCP activity or CDC activity that causes serious side effects, or all It is characterized by not being.
- the present inventors have found for the first time a human CD147 antibody that exhibits antitumor activity by activating cell signaling of CD147 regardless of the effector function of the antibody.
- the function retained by the antibody in the present invention is the binding activity to CD147 and / or the function to activate CD147.
- the antibodies of the invention preferably activate downstream associated signal molecules, such as FAK, MEK, Erk, JAK / STAT, AKT or MAP kinase (MAPK) or downstream of these further downstream associated signal molecules via CD147.
- the antibody of the present invention more preferably activates MAPK or a molecule downstream of MAPK. As MAPK, preferably, p38 MAPK is mentioned.
- Signal molecules further downstream of MAPK include, for example, HSP27, cxcl8 or SMAD (eg, SMAD2, SMAD3 or SMAD4, preferably SMAD4).
- activation of CD147 for example, increase of mRNA expression level of p38 MAPK, increase of protein expression level of p38 MAPK, phosphorylation of p38 MAPK, phosphorylation of HSP27 (for example, phosphorylation of Ser27 of HSP27 or Ser15 of HSP27) Phosphorylation, increase in cxcl8 mRNA expression level, increase in cxcl8 protein expression level, or increase in rhoB mRNA expression level through SMAD signal activation or increase in rhoB protein expression level.
- activation of CD147 preferably, the amount of protein expression of p38 MAPK is increased, the phosphorylation of p38 MAPK, the phosphorylation of HSP27 (eg, the phosphorylation of Ser82 of HSP27 or the phosphorylation of Ser15 of HSP27), the expression of cxcl8 mRNA
- the phosphorylation of p38 MAPK eg, the phosphorylation of Ser82 of HSP27 or the phosphorylation of Ser15 of HSP27
- HSP27 eg, the phosphorylation of Ser82 of HSP27 or the phosphorylation of Ser15 of HSP27
- SMAD2 or SMAD3 is phosphorylated by TGF ⁇ receptor when TGF ⁇ binds to TGF ⁇ receptor (TGFBR1 / 2), forms a heterotrimer with SMAD4, translocates into the nucleus, and SMAD on chromosome It is known that it binds to a transcriptional regulatory region having a DNA binding sequence (Smad binding element: SBE) and positively or negatively regulates mRNA expression of a downstream gene (Miyazono, Nichirei Medical Journal, 1999, 162-166. ). Thus, activation of SMAD4 is considered to require the presence of SMAD2 or SMAD3.
- SMAD2, SMAD3 and SMAD4 negatively regulate the expression level of KLF5 in a TGFb-dependent manner (David et al., Cell, 2016, 164 (5), 1015-1030).
- SMAD4 the suppression signal by SMAD2, SMAD3 and SMAD4 of the KLF5 gene is released, and KLF5 protein is expressed. It is known that SMAD4 is lost and KLF5 expression suppresses TGFb-dependent cell death signal (SOX4 dependent) (supra, David et al, Cell).
- the human CD147 antibody of the invention phosphorylates p38 MAPK (FIG. 21), phosphorylates HSP27 (FIG. 20) and increases the expression of cxcl8 (FIG. 22 (b)) And FIG. 23 (b)). Therefore, the antibody of the present invention can be expressed as CD147 by confirming whether the gene expression or protein expression or phosphorylation state of at least any one of these molecules changes before and after administration of the antibody of the present invention. It can be confirmed whether it is through activation.
- the present inventors show that the human CD147 antibody of the present invention shows efficacy against a pancreatic cancer model in which the SMAD4 protein is expressed (FIG. 25); the human CD147 antibody of the present invention has a gene mutation in SMAD4 In pancreatic cancer models such as BxPC-3 that do not express SMAD4, there are partial (about 30%) antitumor effects (Fig. 24); and administration of the human CD147 antibody of the present invention It was found that the downstream molecules of SMAD2, SMAD3 and SMAD4 (rhoB, FIG. 22 (c) and FIG. 23 (c)) were induced in the tumor after the treatment.
- the antibody of the present invention mediates the activation of CD147 can be confirmed by confirming whether or not the rhoB gene expression or protein expression changes before and after administration of the antibody of the present invention.
- the genomic sequence, gene expression or protein expression of SMAD4 in patient samples is measured using methods well known to those skilled in the art, and a patient expressing SMAD4 is selected as a subject patient to which the antibody of the present invention is to be administered.
- the antibodies of the invention can be administered.
- Liver cancer, ALL, lymphoma, gastrointestinal stromal tumor (GIST), skin cancer, sarcoma, AML, or kidney cancer have a low expression level of KLF5, and it is expected that many patients will be effective for the human CD147 antibody of the present invention.
- gene expression or protein expression of KLF5 in patient samples is measured using a method well known to those skilled in the art, and a patient having reduced or deleted expression of KLF5 is selected as a subject patient to which the antibody of the present invention is to be administered.
- antibodies of the present invention can be administered.
- the degree of reduction in KLF5 expression in this case can be determined by those skilled in the art according to methods known and appropriate clinical trials, for example, the amount of expression of KLF5 in patients who received an effect and patients who did not obtain an effect. And the appropriate threshold is set.
- Fab fragments of antibodies
- F (ab ') 2 Fv, or single chain Fv (scFv)
- diabody diabodies
- Fab ′ which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab ′) 2 under reducing conditions, is also included as a fragment of the antibody.
- the antibodies of the invention may be multispecific antibodies having specificity for at least two different antigens.
- a molecule binds to two types of antigens (ie, bispecific antibodies), but the “multispecific antibodies” in the present invention are more (for example, three types).
- the multispecific antibody of the present invention may be a full-length antibody, or a fragment of such an antibody (eg, F (ab ') 2 bispecific antibody).
- a bispecific antibody can be prepared by combining heavy and light chains (HL pair) of two types of antibodies, or a hybridoma that produces different monoclonal antibodies can be fused to form a bispecific antibody-producing fusion. It can also be produced by producing cells (Millstein et al., Nature (1983) 305, p. 537-539).
- the antibody of the present invention may be a single chain antibody (also described as scFv).
- Single-chain antibodies are obtained by linking the heavy chain variable region of the antibody and the light chain variable region with a polypeptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (eds. Rosenberg and Moore, Springer Verlag, New York, p. 269-315 (1994), Nature Biotechnology (2005), 23, p. 1126-1136)
- a BiscFv fragment prepared by linking two scFvs with a polypeptide linker is used as a bispecific antibody. It can also be used.
- the heavy and light chain variable regions are linked via a non-conjugated linker, preferably a polypeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci.U.S.A. (1988), 85, p. 5879-5883).
- the heavy and light chain variable regions in the scFv may be from the same antibody or from separate antibodies. For example, any single-chain peptide consisting of 12 to 19 residues is used as a polypeptide linker for linking variable regions.
- the DNA encoding the scFv is a DNA encoding the heavy chain or heavy chain variable region of the antibody, and a DNA encoding the light chain or light chain variable region, all or desired amino acid sequence of those sequences
- the DNA fragment to be encoded is used as a template and amplified by PCR using a primer pair that defines both ends, and then DNA encoding the polypeptide linker moiety is further ligated to heavy chain and light chain respectively. It is obtained by combining and amplifying the primer pair prescribed in.
- an expression vector containing them and a host transformed with the expression vector can be obtained according to a conventional method, and by using the host, ScFv can be obtained according to the method.
- ScFv can be obtained according to the method.
- the antibodies of the present invention may be multimerized to increase the affinity for the antigen.
- the antibody to be multimerized may be a single antibody or multiple antibodies that recognize multiple epitopes of the same antigen. Examples of methods for multimerizing antibodies include binding of an IgG CH3 domain to two scFvs, binding to streptavidin, introduction of a helix-turn-helix motif, and the like.
- the antibody of the present invention may be a polyclonal antibody, which is a mixture of multiple types of anti-CD147 antibodies differing in amino acid sequence.
- a polyclonal antibody a mixture of multiple types of antibodies with different CDRs can be mentioned.
- a polyclonal antibody a mixture of cells producing different antibodies can be cultured, and an antibody purified from the culture can be used (see WO 2004/061104).
- the antibody of the present invention may be an antibody having 80% to 99% identity (or homology) as compared to the heavy chain and / or light chain of the above-mentioned antibody.
- identity or homology
- the antibody of the present invention may be an antibody having 80% to 99% identity (or homology) as compared to the heavy chain and / or light chain of the above-mentioned antibody.
- amino acid sequences of heavy chain and / or light chain with one or more amino acid residues substituted, deleted and / or added, various actions equivalent to the above-mentioned respective antibodies can be obtained. It is possible to select the antibody that it has.
- the number of amino acid residues to be substituted, deleted and / or added is generally 10 amino acid residues or less, preferably 5 to 6 amino acid residues or less, more preferably 2 to 3 amino acid residues or less Most preferably one amino acid residue.
- the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in mammalian cultured cells is deleted (Tsubaki et. Al., Int. J. Biol. Macromol, 139-147, 2013).
- the present invention also includes the antibody that receives the modification, a deletion form in which one or two amino acids are deleted at the heavy chain carboxyl terminus, and a deletion form that is amidated (for example, a carboxyl terminal site Heavy chain in which a proline residue is amidated) and the like can be mentioned.
- the deletion of the carboxyl terminus of the heavy chain of the antibody according to the present invention is not limited to the above-mentioned type, as long as the antigen binding ability and the function to activate related signal molecules downstream of CD147 are maintained.
- the two heavy chains constituting the antibody according to the present invention may be any one kind of heavy chain selected from the group consisting of full length and the above-mentioned deletion product, or any two kinds thereof are combined. It may be one.
- the quantitative ratio of each deletion body may be influenced by the type and culture conditions of mammalian cultured cells producing the antibody according to the present invention, both main chains of the antibody according to the present invention may be carboxyl in both of two heavy chains. There may be mentioned the case where one terminal amino acid residue is deleted.
- Blast algorithm version 2.2.2 Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402).
- Blast algorithm is available on the Internet at www. ncbi. nlm. nih. It can also be used by accessing gov / blast.
- the value of two types of percentages of Identity (or Identities) and Positivity (or Posibilities) is calculated by the above-mentioned Blast algorithm.
- the former is the value when the amino acid residue is identical between the two types of amino acid sequences to be determined for homology
- the latter is a numerical value taking into account the amino acid residue having a similar chemical structure.
- the value of Identity (identity) when amino acid residues are identical is taken as the value of homology.
- antibodies conjugated to various molecules such as polyethylene glycol (PEG) can also be used.
- PEG polyethylene glycol
- the antibody of the present invention may further be one in which these antibodies and another agent form a conjugate (immunoconjugate).
- conjugate examples include those in which the antibody is linked to a radioactive substance or a compound having pharmacological activity (Nature Biotechnology (2005) 23, p. 1137-1146).
- the resulting antibodies can be purified to homogeneity.
- separation and purification methods used for ordinary proteins may be used.
- antibodies can be separated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, polyacrylamide gel electrophoresis for preparation, isoelectric focusing electrophoresis etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory ( 988)) it is not intended to be limited thereto.
- chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography and the like.
- chromatographies can be performed using liquid chromatographies, such as HPLC and FPLC.
- the anti-CD147 antibody of the present invention can be obtained from among the anti-CD147 antibodies obtained by the method described in the above-mentioned “preparation of anti-CD147 antibody”. The antibody thus obtained can be used as a therapeutic and / or prophylactic agent for tumors and / or cancers.
- the anti-CD147 antibody of the present invention has excellent antitumor activity and is useful as a therapeutic agent for tumor or cancer.
- the anti-CD147 antibody of the present invention also showed excellent antitumor effect against gemcitabine-resistant cancer cells and sorafenib low-sensitivity cancer cells.
- the anti-CD147 antibody of the present invention showed remarkable efficacy in chronic myelogenous leukemia cells than imatinib.
- Tumors which can be treated by the anti-CD147 antibody of the present invention or a drug containing the antibody are not particularly limited as long as they are tumors expressing CD147, but preferably pancreatic cancer, liver cancer, gastric cancer, colon cancer, renal cancer, Breast cancer, uterine cancer, ovarian cancer, lung cancer, thyroid cancer, skin cancer, head and neck cancer, sarcoma, prostate cancer, bladder cancer, brain tumor, gastrointestinal stromal tumor (GIST), leukemia (eg, acute myeloid leukemia (AML) Chronic myelogenous leukemia (CML), or chronic lymphocytic leukemia (CLL) or acute lymphocytic leukemia (ALL), lymphoma or malignant lymphoma (eg, B cell lymphoma, non-Hodgkin's lymphoma or diffuse large cell B cell lymphoma) (Diffuse large B-cell lymphoma, DLBCL)), more preferably pancreatic cancer, liver cancer, gastric cancer, large Cancer,
- tumors that can be treated by the antibody of the present invention or a drug containing the antibody include tumors that express SMAD-positive CD147, and examples of tumors that express SMAD-positive CD147 include, for example, SMAD-positive CD147.
- Liver cancer or pancreatic cancer that expresses The anti-CD147 antibody of the present invention or a drug containing the antibody is preferably administered to a patient whose expression of CD147 and / or SMAD has been confirmed.
- SMAD Preferably, SMAD2, SMAD3 and / or SMAD4 are mentioned, More preferably, SMAD4 is mentioned.
- it is confirmed that at least any one of SMAD2 or SMAD3 is expressed in accordance with the confirmation of the expression of SMAD4.
- a tumor that can be treated by the antibody of the present invention or a drug containing the antibody includes a tumor in which expression of KLF5 is reduced or deleted, and a tumor in which expression of KLF5 is reduced or deleted is liver cancer, These include ALL, lymphoma, gastrointestinal stromal tumor (GIST), skin cancer, sarcoma, AML or kidney cancer.
- the anti-CD147 antibody of the present invention or a drug containing the antibody is preferably administered to a patient for whom expression of KLF5 has been confirmed to be reduced or deleted.
- the anti-CD147 antibody of the present invention can also be administered two, three or more other therapeutic agents depending on the therapeutic purpose, and these other therapeutic agents can be simultaneously administered by encapsulating them in the same formulation. can do.
- the other therapeutic agent and the anti-CD147 antibody can also be administered simultaneously by encapsulating them in the same formulation.
- the anti-CD147 antibody and the other therapeutic agent can be enclosed in separate formulations and administered simultaneously.
- the other agent and the anti-CD147 antibody can be administered separately in tandem.
- a therapeutic agent containing an anti-CD147 antibody or an antigen-binding fragment of the antibody as an active ingredient is administered, or an anti-CD147 antibody or an antigen-binding fragment of the antibody is contained as an active ingredient
- Other therapeutic agents may be administered after administering the therapeutic agent.
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising a therapeutically and / or prophylactically effective amount of an anti-CD147 antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.
- the present invention comprises a therapeutically and / or prophylactically effective amount of an anti-CD147 antibody and a therapeutically and / or prophylactically effective amount of at least one antitumor therapeutic agent and a pharmaceutically acceptable diluent, carrier, solubilizer, Also provided is a pharmaceutical composition comprising an emulsifier, a preservative and / or an adjuvant.
- the substance to be used for the acceptable formulation in the pharmaceutical composition of the present invention is preferably one that is nontoxic to the person to whom the pharmaceutical composition is administered, preferably at the dosage and concentration.
- the pharmaceutical composition of the present invention changes or holds pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, dissolution rate, sustained release rate, absorption rate, permeability. It can contain substances for formulation of The substances for preparation may include, but are not limited to: amino acids such as glycine, alanine, glutamine, asparagine, arginine or lysine, antibacterial agents, ascorbic acid, sodium sulfate or sodium bisulfite, etc.
- Antioxidants buffers such as phosphoric acid, citric acid, borate buffer, sodium hydrogen carbonate, tris-hydrochloric acid (Tris-Hcl) solution, fillers such as mannitol and glycine, chelating agents such as ethylenediaminetetraacetic acid (EDTA) , Caffeine, polyvinyl pyrrolidine, complexing agents such as ⁇ -cyclodextrin and hydroxypropyl- ⁇ -cyclodextrin, extenders such as glucose, mannose or dextrin, other carbohydrates such as monosaccharides and disaccharides, coloring agents, flavors Agents, diluents, emulsifiers, polyvinyl pyrrolidine etc.
- Tris-Hcl tris-hydrochloric acid
- Hydrophilic polymer low molecular weight polypeptide, salt forming counter ion, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorexidine, chloric acid, sorbic acid or hydrogen peroxide etc., glycerin, propylene -Solvents such as glycol or polyethylene glycol, sugar alcohols such as mannitol or sorbitol, suspensions, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, etc., surface activity such as triton, tromethamine, lecithin or cholesterol Agents, stabilization enhancers such as sucrose and sorbitol, elasticity enhancers such as sodium chloride, potassium chloride and mannitol / sorbitol, transport agents, excipients, / Or pharmaceutically adjuvants.
- glycerin propylene -So
- the addition amount of the substance for these preparations is preferably 0.01 to 100 times, particularly 0.1 to 10 times the weight of the anti-CD147 antibody.
- the composition of a suitable pharmaceutical composition in a preparation can be appropriately determined by those skilled in the art according to the disease to be applied, the administration route of application, and the like.
- the excipient or carrier in the pharmaceutical composition may be liquid or solid. Suitable excipients and carriers may be water or saline for injection, artificial cerebrospinal fluid, or other substances commonly used for parenteral administration. Neutral saline or saline containing serum albumin can also be used as a carrier.
- the pharmaceutical composition can include Tris buffer of pH 7.0-8.5, acetate buffer of pH 4.0-5.5, citrate buffer of pH 3.0-6.2. These buffers can also include sorbitol and other compounds.
- the pharmaceutical composition of the present invention can include a pharmaceutical composition containing an anti-CD147 antibody, and a pharmaceutical composition containing an anti-CD147 antibody and at least one antitumor therapeutic agent, and the pharmaceutical composition of the present invention is selected.
- compositions comprising an anti-CD147 antibody, and pharmaceutical compositions comprising an anti-CD147 antibody and at least one anti-cancer drug therapeutic agent can also be formulated as a lyophilizate using appropriate excipients such as sucrose.
- the pharmaceutical composition of the present invention can be prepared for parenteral administration or can be prepared for oral gastrointestinal absorption.
- the composition and concentration of the formulation can be determined by the method of administration.
- the antibody of the present invention is administered to humans, about 0.1 to 100 mg / kg may be administered one or more times in 1 to 180 days.
- the dose and the number of doses should generally be determined in consideration of the patient's sex, weight, age, symptoms, severity, side effects, etc., they are not limited to the above dose or usage. I assume.
- Examples of the form of the pharmaceutical composition of the present invention include an injection containing a drop, a suppository, a nasal agent, a sublingual agent, a percutaneous absorption agent and the like.
- the administration route is an oral route or a parenteral route, and a parenteral route includes, for example, intravenous, intraarterial, intramuscular, intrarectal, transmucosal, intradermal and the like.
- the antibody of the present invention or an antigen-binding fragment of the antibody, a drug complex containing them, a bispecific antibody containing them or a pharmaceutical composition containing them in combination with a biomarker for screening patients to which they are administered can be provided.
- These antibodies and pharmaceutical compositions may be provided as a kit in combination with a means for detecting a biomarker, and provision of an antibody or pharmaceutical composition and provision of a biomarker may be separate.
- the antibody or pharmaceutical composition of the present invention can be administered to a group of patients in which the effect of the antibody of the present invention is expected to be higher.
- the present invention measures the expression of SMAD4 or the expression of KLF5 contained in a biological sample using a biological sample derived from a cancer patient, and the expression reduction or deletion of the patient in which SMAD4 is detected or KLF5 Predicting responsiveness to treatment of cancer, including determining the detected patient as being responsive to treatment of cancer with the antibody of the present invention or a functional fragment of the antibody or the pharmaceutical composition of the present invention
- a biological sample derived from a cancer patient was used to detect the expression of SMAD4 or KLF5 in the biological sample, and the expression reduction or deletion of the patient in which SMAD4 was detected or KLF5 was detected
- a method of screening a subject for treating cancer comprising screening a patient as a subject of treating cancer with the antibody of the present invention or a functional fragment of the antibody or the pharmaceutical composition of the present invention; For a patient in which SMAD4 expression or KLF5 expression contained in the biological sample is detected and a SMAD4 detected or a KLF5 expression reduction or deletion is detected, using
- biological sample refers to tissue, fluid, cells, and mixtures thereof isolated from an individual, such as a tumor biopsy, spinal fluid, intrathoracic fluid, intraperitoneal fluid, lymph fluid, skin A section, blood, urine, feces, sputum, respiratory tract, intestinal tract, urogenital tract, saliva, milk, digestive organs and cells collected therefrom can be mentioned, but it is not limited thereto.
- the “biological sample” is preferably a sample containing a cancer cell, more preferably a tissue or cell obtained by excision or biopsy, or a cell derived from an intrathoracic fluid or an intraperitoneal fluid can be exemplified. . Further preferred biological samples are samples containing cancer cells or cancer tissues.
- SMAD4 The detection and measurement of “expression of SMAD4” can be carried out using methods known to those skilled in the art for genomic sequence, gene expression or protein expression of SMAD4, and examples include RNA sequencing, microarray, genomic sequencing, and immunoassays.
- the detection and measurement of "expression of KLF5" can detect the genomic sequence, gene expression or protein expression of KLF5 using methods well known to those skilled in the art, for example, IHC, RNA sequencing, microarray, genomic sequencing, immunoassay Can be mentioned.
- the “reduced expression of KLF5” means that the expression level is lower in comparison with a control (eg, the expression level in non-cancer tissues of healthy individuals and the same patient).
- the degree of reduction in KLF5 expression that can be determined to be responsive to treatment of cancer with the antibody or pharmaceutical composition of the present invention is determined by those skilled in the art by performing known methods and appropriate clinical tests.
- An appropriate threshold value is set by, for example, comparing the expression level of KLF5 in a patient who has obtained an effect and a patient who has not obtained an effect. Therefore, "a reduction in the expression of KLF5" means, for example, below the threshold set in this way.
- Example 1 Preparation of Mouse-Rat Antibody by Cell Immunization 1
- CD147 Expression Vector Clone IOH 3378 (Invitrogen) of a commercially available human CD147 gene (BSG variant 2 / CD 147 v 2) and an expression vector pcDNA-DEST 40 for mammalian cells (Invitrogen) was reacted with Gateway LR clonase to produce a human CD147v2 expression vector (pcDNA-DEST40-CD147v2).
- a commercially available expression vector pCMV6-XL5-hBSGv1 (Origene, Cat. SC303059) for mammalian cells of a commercially available human CD147 gene (BSG variant1 / CD147v1) was purchased and used as a human CD147v1 expression vector.
- PCMV3-cynoBSG (Sino Biological Inc., Cat. CG90636-UT) was purchased as a cynomolgus monkey CD147 expression vector.
- PCMV3-mBSGv2 (Sino Biological Inc., Cat. MG 50332-UT) was purchased as a mouse CD147v2 expression vector.
- ClonaCell-HY Medium D (STEMCELL TECHNOLOGIES)
- ClonaCell-HY Medium E (STEMCELL TECHNOLOGIES) were used.
- Human CD147 Fc fusion protein (Sino Biological, Cat. 10186-H02H) and mouse CD147 Fc fusion protein (Sino Biological, Cat. 50332-M03H) were used.
- Human CD147 Fc fusion protein and mouse CD147 Fc fusion protein were added to PBS buffer, dissolved on ice, and prepared to 1 ⁇ g / ml. 100 ⁇ l of the same protein solution was added to a 96-well plate (NUNC, Cat. 442404), stored at 4 ° C. overnight, and the wells were coated with a CD147 Fc fusion protein. The protein lysate was removed and the wells were blocked for 2 hours at 4 ° C.
- Example 1 Preparation of monoclonal antibody and determination of antibody isotype
- Example 1 With respect to the hybridomas for which anti-human CD147 antibody production has been confirmed in Example 4 and which can be stably cultured, culturing is performed using a commercially available Isotyping kit The isotype of the antibody contained in the supernatant was determined and is shown in Table 2. These hybridomas were cultured using CL-1000 flask (Nippon Becton Dickinson Co., Ltd.) to prepare a hybridoma culture supernatant containing a monoclonal antibody.
- the antibody was purified from the culture supernatant prepared in Example 1) -5.
- the anti-human CD147 mouse monoclonal antibody was purified in one step with rProtein A affinity chromatography (at 4 to 6 ° C.).
- the buffer displacement step after rProtein A affinity chromatography purification was performed at 4 to 6 ° C.
- the culture supernatant was applied to a column packed with PBS equilibrated MabSelectSuRe (manufactured by GE Healthcare Bioscience). After all of the culture solution entered the column, the column was washed with a column volume of twice or more of PBS.
- the anti-human CD147 rat monoclonal antibody was purified in one step of Protein G affinity chromatography (at 4 to 6 ° C.).
- the buffer displacement step after protein G affinity chromatography purification was performed at 4 to 6 ° C.
- hybridoma culture supernatant was applied to a column packed with Protein G (GE Healthcare Bioscience) equilibrated with PBS. After all of the culture supernatant fluid entered the column, the column was washed with PBS having a column volume of twice or more. Then, it was eluted with 0.1 M aqueous glycine / hydrochloric acid solution (pH 2.7), and fractions containing the antibody were collected.
- the collected fraction was adjusted to pH 7.0-7.5 by adding 1 M Tris-HCl (pH 9.0), and then added to Centrifugal UF Filter Device VIVASPIN 20 (molecular weight cut UF 30 K, Sartorius, 4 to 6 ° C.). Then, the buffer was replaced with HBSor (25 mM Histidine / 5% Sorbitol / pH 6.0) and concentrated to adjust the antibody concentration to 1 mg / mL or more. Finally, the resultant was filtered with Minisart-Plus filter (Sartorius) to obtain a purified sample.
- HBSor 25 mM Histidine / 5% Sorbitol / pH 6.0
- the major axis and minor axis of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated by the following formula.
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in FIGS. 1 (a) to (c).
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- the results of the 2P2D6 antibody, 2P3G8 antibody and 2P2D10 antibody are shown in FIG. 1 (a).
- the tumor growth inhibition rate at 48 days after transplantation which is the final measurement day was 10%, 45% and 40% in the 10 mg / kg administration group, respectively.
- the results of the LN22R8 antibody, 2P1A6 antibody and 2P1B7 antibody are shown in FIG. 1 (b).
- the tumor growth inhibition rate at 48 days after transplantation which is the final measurement day was 50%, 26% and 24% in the 10 mg / kg administration group, respectively.
- the results of the 2P8C12 antibody and 2P10F2 antibody are shown in FIG. 1 (c).
- the tumor growth inhibition rate at 48 days after transplantation, which is the final measurement date, was -2% and 62% in the 10 mg / kg administration group, respectively.
- Example 1 1) -8 Analysis of species cross-reactivity of CD147 antibody It was prepared in Example 1) -1 using Lipofectamine 2000 (Thermofishers Scientific, Cat. 11668-019) in CHO-K1 cells (ATCC, CCL-61). One day after introducing pcDNA-DEST40-CD147v2 or pCMV3-cynoBSG, mouse anti-human CD147 antibody (LN22R8) or rat anti-human CD147 antibody (2P1A6, 2P1B7, 2P3G8, 2P2D10, 2P8C12, 2P10F2, 2P2D6), 10 ⁇ g / ⁇ g treated with anti-mouse IgG-FITC (MP Biomedical, Cat.
- anti-CD147 antibody MEM-M6 / 1
- mouse anti-human CD147 antibody LN22R8
- rat anti-CD147 antibody 2P1A6, 2P1B7, 2P3G8, 2P2D10, 2P8C12, 2P10F2, 2P2D6
- any anti-CD147 antibody is also human CD147 It showed binding to expressing CHO-K1 cells (FIGS. 2-1 to 3).
- the commercially available anti-CD147 antibody (MEM-M6 / 1) showed binding to cynomolgus monkey CD147-expressing CHO-K1 cells, mouse anti-human CD147 antibody (LN22R8), rat anti-human CD147 antibody (2P1A6, 2P1B7, 2P3G8, 2P2D10) , 2P8C12, 2P10F2 and 2P2D6), none of the CD147 antibodies showed binding to cynomolgus monkey CD147-expressing CHO-K1 cells (FIGS. 2-1 to 3).
- the amino acid sequence contained in common to hCD147v1 and v2 was subjected to sequence comparison in cynomolgus monkey and human CD147, and classified into 9 regions of amino acid regions different between species, and designated as mu1 to mu9 (FIG. 3).
- the cDNA sequence of human CD147 variant 2 into which a FLAG sequence was introduced at the N-terminus was artificially synthesized for preparation of the CD147 mutant expression vector and expression confirmation on the cell membrane, and the plasmid introduced into pcDNA3.1 vector, Signal-N-Flag-hCD147 v2_pcDNA3. 1 (made by Genscript) was prepared.
- hCD147-mu1_pcDNA3.1 9 kinds of human-cynomolgus monkey CD147 expression vectors introduced as amino acid substitution mutations by DNA substitution into the CD147 amino acid sequence mu1 to mu9 of cynomolgus monkey in the human CD147 gene of the same plasmid, hCD147-mu1_pcDNA3.1, hCD147-mu2_pcDNA3.1, hCD147 -Mu3_pcDNA3.1, hCD147-mu4_pcDNA3.1, hCD147-mu5_pcDNA3.1, hCD147-mu6_pcDNA3.1, hCD147-mu7_pcDNA3.1, hCD147-mu8_pcDNA3.1, hCD147-mu9_pcDNA3.1 (prepared by Genscript).
- Example 1 The antibodies 2P3G8, 2P10F2, 2P2D10 and LN22R8 in which an antitumor effect of 40% or more was observed in Example 7) all lost the binding to CD147 having a mutation of mu3. It was suggested that the epitope important for the antitumor effect is the m3 region.
- RNA of Production Hybridoma In order to amplify cDNA encoding the variable region of LN22R8 antibody, total RNA was prepared from LN22R8 antibody-producing hybridoma using TRIzol Reagent (Ambion).
- CDNA encoding the variable region of the light chain amplified by 5'-RACE PCR was cloned into a plasmid, and then sequence analysis of the nucleotide sequence of the cDNA encoding the variable region of the light chain was performed.
- the nucleotide sequence of the cDNA encoding the variable region of the light chain of LN22R8 antibody determined is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.
- the CDRL1, CDRL2 and CDRL3 of the light chain variable region of the LN22R8 antibody are shown in SEQ ID NOs: 11, 12 and 13, respectively.
- CDNA encoding the variable region of heavy chain amplified by 5'-RACE PCR was cloned into a plasmid, and then sequence analysis of the nucleotide sequence of cDNA encoding variable region of heavy chain was performed.
- the nucleotide sequence of the cDNA encoding the variable region of the heavy chain of LN22R8 antibody determined is shown in SEQ ID NO: 9 and the amino acid sequence is shown in SEQ ID NO: 10.
- the CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the LN22R8 antibody are shown in SEQ ID NOs: 14, 15 and 16, respectively.
- the nucleotide sequence of the cDNA encoding the variable region of the light chain of the 2P10F2 antibody determined is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18.
- the CDRL1, CDRL2 and CDRL3 of the light chain variable region of the 2P10F2 antibody are shown in SEQ ID NOs: 21, 22 and 23, respectively.
- the nucleotide sequence of the cDNA encoding the variable region of the heavy chain is shown in SEQ ID NO: 19 and the amino acid sequence is shown in SEQ ID NO: 20.
- the CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the 2P10F2 antibody are shown in SEQ ID NOs: 24, 25 and 26, respectively.
- PCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3 / LK.
- IgG1 type heavy chain expression vector of human chimeric LN22R8 1) Designed for in-fusion cloning using cDNA encoding the variable region of LN22R8 heavy chain obtained in 11-11-1-3 as a template
- the DNA fragment containing the cDNA encoding the variable region of the heavy chain was amplified by performing PCR with the above primers.
- the amplified DNA fragment was inserted at the site where pCMA-G1 was cleaved with restriction enzyme BlpI, to construct an IgG1 type heavy chain expression vector of human chimeric LN22R8.
- the nucleotide sequence of the IgG1 type heavy chain of human chimeric LN 22 R8 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 32 and SEQ ID NO: 33, respectively.
- Example 1 -13-3 Construction of Light Chain Expression Vector of Human Chimera 2P10F2
- Example 1 Using the cDNA encoding the variable region of the light chain of 2P10F2 obtained in Example 1-11-2 as a template, Example 1-12 A light chain expression vector of human chimera 2P10F2 was constructed in the same manner as -4.
- the nucleotide sequence of the light chain of human chimeric 2P10F2 and the amino acid sequence of the light chain are shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively.
- Example 1 1) -13-5 Construction of IgG2 type heavy chain expression vector of human chimera 2P10F2
- Example 1 Using the cDNA encoding the variable region of the heavy chain of 2P10F2 obtained in Example 1-11-2 as a template
- An IgG2-type heavy chain expression vector of human chimera 2P10F2 was constructed in the same manner as in Example 12-12.
- the nucleotide sequence of the IgG2 type heavy chain of human chimeric 2P10F2 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 44 and SEQ ID NO: 45, respectively.
- the human chimeric antibody of LN22R8 obtained by combining the IgG1 type heavy chain expression vector of human chimeric LN22R8 and the light chain expression vector of human chimeric LN22R8 was named “LN22R8 chIgG1”.
- the human chimeric antibody of LN22R8 obtained by combining the IgG2 type heavy chain expression vector of human chimeric LN22R8 and the light chain expression vector of human chimeric LN22R8 was named “LN22R8 chIgG2”.
- a human chimeric antibody of LN22R8 obtained by combining an IgG4P-type heavy chain expression vector of human chimeric LN22R8 and a light chain expression vector of human chimeric LN22R8 was named “LN22R8 chIgG4P”.
- the L2P10F2 human chimeric antibody obtained by combining the human chimeric 2P10F2 IgG1 LALA type heavy chain expression vector and the human chimeric 2P10F2 light chain expression vector was designated as "2P10F2 chIgG1 LALA”.
- a human chimeric antibody of L2P10F2 obtained by combining an IgG2 type heavy chain expression vector of human chimeric 2P10F2 and a light chain expression vector of human chimeric 2P10F2 was named “2P10F2chIgG2”.
- a human chimeric antibody of L2P10F2 obtained by combining an IgG4P-type heavy chain expression vector of human chimeric 2P10F2 and a light chain expression vector of human chimeric 2P10F2 was named “2P10F2chIgG4P”.
- Example 1 Purification of Human Chimeric Antibody of LN22R8, 2P10F2
- the antibody was purified from the culture supernatant obtained in Example 1) -14-1 by a one-step process of rProtein A affinity chromatography.
- the culture supernatant was applied to a column (manufactured by GE Healthcare Bioscience) packed with PBS equilibrated with MabSelectSuRe, and then the column was washed with 2 column volumes or more of PBS. Then, it was eluted with 2 M arginine hydrochloride solution (pH 4.0), and fractions containing the antibody were collected.
- 2 M arginine hydrochloride solution pH 4.0
- the fraction was subjected to buffer substitution to HBSor (25 mM histidine / 5% sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette).
- the antibody was concentrated with Centrifugal UF Filter Device VIVASPIN 20 (fractional molecular weight UF10K, Sartorius) to adjust the IgG concentration to 1 mg / mL or more.
- the resultant was filtered with Minisart-Plus filter (Sartorius) to obtain a purified sample.
- ADCC activity of Human Chimeric Antibody was evaluated using human peripheral blood mononuclear cells (PBMC) as effector cells and human pancreatic strain MIA PaCa-2 as ADCC target cells. Radioisotopes 51 Cr labeled with MIA PaCa-2 cells and mouse antibody (LN22R8), rat antibody (2P10F2), or human chimeric antibody (LN22R8chIgG1, LN22R8chIgG2, LN22R8chIgG4P, 2P10F2chIgG1LALA , 2P10F2chIgG4P) 0.5 or 5 [mu] g / ml After treatment at 4 ° C.
- PBMC peripheral blood mononuclear cells
- the measurement was similarly performed on a sample treated with human IgG (hIgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) as a negative control sample, and is shown together.
- the measurement was performed in triplicate, and the mean value and the standard deviation were calculated and shown together.
- % Specific release (Total release-Spontaneous release) / Maximamu release Human IgG (hIgG) and LN22R8 mouse antibodies did not show ADCC activity, whereas LN22R8 chIgG1 showed ADCC activity with 17.4% at 0.5 ⁇ g / ml and 18.1% at 5 ⁇ g / ml.
- the ADCC activities of LN22R8 chIgG2 and LN22R8 chIgG4P were lower than that of LN22R8 chIgG1, and even at 5 ⁇ g / ml, they were 3.0% and 2.2%, respectively.
- the 2P10F2 rat antibody exhibited ADCC activity of 4.8% at 0.5 ⁇ g / ml and 8.4% at 5 ⁇ g / ml.
- 2P10F2ch IgG1 LALA showed ADCC activity as 4.7% at 0.5 ⁇ g / ml and 2.9% at 5 ⁇ g / ml.
- 2P10F2chIgG4P showed lower ADCC activity than 3.4P at 0.5 ⁇ g / ml, 1.1% at 5 ⁇ g / ml and 2P10F2 rat antibody, 2P10F2 chIgG1LALA.
- the human chimeric antibody using the IgG1 subtype showed the highest ADCC activity.
- CDC activity The complement-dependent cell killing activity (CDC activity) by the anti-human CD147 antibody was evaluated using human pancreatic strain MIA PaCa-2 as a target cell.
- a commercially available rabbit complement (Low Tox-M Rabbit Complement, CEDARLANE LABORATORIES LIMITED, Cat. CL3051) was used as a complement.
- an anti-human CD147 antibody a mouse antibody (LN22R8) or a rat antibody (2P10F2) or a human chimeric antibody (LN22R8chIgG1, LN22R8chIgG2, LN22R8chIgG4P, 2P10F2chIgG1LALA, 2P10F2chIgG4P) was used.
- Human IgG (hIgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) was used as a CDC activity negative control antibody. After treating the above antibody at a concentration of 0, 0.1, 1 or 10 ⁇ g / ml for 1 hour at 4 ° C, add rabbit complement to a final concentration of 7.5%, 37 ° C, 5% After warming for 3 hours in the presence of CO 2, intracellular ATP contained in living cells was measured using CellTiter-Glo Lumimescent Cell Viability Assay (Promega, Cat. G7572).
- the luminescence signal obtained using the CellTiter-Glo Lumimescent Cell Viability Assay was quantified using EnVision 2104 Multilabel Reader (Perkin Elmer). The measurement was performed in triplicate, and the mean value and the standard deviation were calculated. The luminescence signal obtained from untreated cells was taken as 100%, and the luminescence signal that decreased depending on the antibody and complement was shown as CDC activity in FIG.
- Concentration-dependent CDC activity was observed only for the mouse antibody (LN22R8) and rat antibody (2P10F2) relative to hIgG as a negative control, and LN22R8 reduced viable cells up to 41.1% at 10 ⁇ g / ml. In 2P10F2, viable cells were reduced up to 53.5% at 10 ⁇ g / ml.
- ADCP activity of human chimeric antibody Human IgG antibody induces antibody-dependent phagocytosis (ADCP) by monocytes and macrophages via interaction with mouse Fc ⁇ receptor, thereby acting on cancer cells. It has been reported to exhibit cell killing activity (Overdijk et al., Journal of Immunology, 1-9, 2012).
- the ADCP activity of human chimeric antibodies was evaluated using RAW 264.7 (ATCC, TIB-71) as effector cells and human pancreatic strains PANC-1 or MIA PaCa-2 as ADCP target cells.
- PKH67 Green Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling SIGMA, Cat.
- the measurement was similarly performed on a sample treated with human IgG (hIgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) as a negative control sample.
- the measurement was performed in triplicate, the average value and the standard deviation were calculated, and the result of FIG. 8 (a) PANC-1 was shown, and the result of FIG. 8 (b) MIA PaCa-2 was shown.
- LN22R8 chIgG1 When PANC-1 cells were targeted for ADCP, LN22R8 chIgG1 showed 9.2% and LN22R8 chIgG4P showed 9.0% higher ADCP activity than human IgG (5.5%). LN22R8 chIgG2 was 5.9% and was negative for ADCP activity.
- LN22R8chIgG1 has 6.6%
- LN22R8chIgG4P has 6.1% higher ADCP activity than human IgG (3.6%).
- LN22R8 chIgG2 was 3.6% and was negative for ADCP activity.
- the major axis and minor axis of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated by the following formula.
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in FIGS. 9-1 (a) to (d) and 9-2 (e) to (g).
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- LN22R8 showed a mouse antibody and three human chimeric antibodies also have dose-dependent antitumor effects.
- LN22R8chIgG4P 10 mg / kg administration group complete regression of tumors was observed in 5 mice out of 5 18 days after transplantation, and no tumor regrowth was observed 41 days after transplantation at the end of the experiment.
- tumor regrowth was observed in some or all mice.
- 2P10F2 was confirmed to have an antitumor effect with a rat antibody and two human chimeric antibodies.
- 2P10F2ch IgG4P 10 mg / kg administration group complete regression of tumors was observed in 6 mice out of 6 21 days after transplantation.
- mouse antibody LN22R8 and rat antibody 2P10F2 which are anti-human CD147 antibodies that recognize the same epitob moiety, have human chimeric antibody chIgG1 having effector function dependent on mouse immune system such as ADCC activity, ADCP and CDC activity, and human chimeric antibody chIgG4P having ADCP activity
- human chimeric antibody chIgG2 which hardly shows any effector function, has 90% or more of the anti-tumor effect, so it does not depend on mouse immunity and has a novel mechanism of action acting on CD147. It was suggested that the tumor effect was shown.
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in FIG.
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- the tumor growth inhibition rate at 17 days after transplantation was 99% in the 10 mg / kg administration group, and complete regression of the tumor was observed in 3 out of 5 mice.
- Anti-CD147 human chimeric antibody (LN22R8chIgG4P) showed strong antitumor effect against pancreatic cancer tumors formed in NK cells and NOG mice lacking complement activity, in addition to mouse T and B cells It has been suggested that it may have an anti-tumor effect independent of immune cells in mice.
- Example 2 Preparation of Monkey Crossing Rat Antibody by Immunization with CD147 Protein
- the anti-human CD147 antibody showing strong antitumor effect obtained in Example 1 showed no cross reactivity to mouse, rat or cynomolgus monkey CD147 .
- 2P10F2 obtained in Example 1 showed binding to MIA PaCa-2, CHO-K1-hCD147v2 and showed no binding to CHO-K1-cynoCD147.
- rat_CD147_ # 84 (sometimes referred to herein as r # 84), rat_CD147_ # 131 (sometimes referred to herein as r # 131), rat_CD147_ # 110 (sometimes referred to herein as r # 110) and rat_CD147_ # 101 (sometimes referred to as r # 101 in the present specification) bind to MIA PaCa-2, CHO-K1-hCD147v2, and CHO-K1-cynoCD147.
- An anti-human CD147 rat antibody showing cynomolgus monkey cross-reactivity was obtained.
- Example 2) -6 Purification of Monoclonal Antibody
- the antibody was purified from the culture supernatant prepared in Example 2) -4 and Example 2) -5 in the same manner as in Example 1) -6.
- the mice in the control group were similarly administered intraperitoneally with PBS.
- Six days after the cynomolgus monkey cross-linking anti-CD147 rat antibody # 131 was implanted, 10 mg / kg was intraperitoneally administered to tumor-bearing mice (n 5).
- the major axis and minor axis of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated by the following formula.
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in FIGS. 11 (a) to (d).
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- the tumor growth inhibition rate at 28 days after transplantation was 95% in the 10 mg / kg administration group (FIG. 11 (a)).
- r # 101 had a tumor growth inhibition rate of 37% in the 10 mg / kg administration group 15 days after transplantation, tumor regrowth was observed 28 days after transplantation (FIG. 11 (b)).
- r # 110 had a tumor growth inhibition rate of 51% after 10 days of transplantation in the 10 mg / kg administration group, tumor regrowth was observed 28 days after transplantation (FIG. 11 (c)).
- the inhibition rate of tumor growth of r # 131 16 days after transplantation was 50% in the 10 mg / kg administration group (FIG. 11 (d)).
- a rat antibody strongly inhibiting tumor growth of MIA PaCa-2, r # 84, and partially inhibiting r # 101, r # 110, r # 131 were obtained.
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- r # 84 had a tumor growth inhibition rate of 95% in the 10 mg / kg administration group 28 days after transplantation (FIG. 11 (a)).
- r # 101 had a tumor growth inhibition rate of 37% in the 10 mg / kg administration group 15 days after transplantation, tumor regrowth was observed 28 days after transplantation (FIG. 11 (b)).
- r # 110 had a tumor growth inhibition rate of 51% after 10 days of transplantation in the 10 mg / kg administration group, tumor regrowth was observed 28 days after transplantation (FIG. 11 (c)).
- the tumor growth inhibition rate at 16 days after transplantation was 50% in the 10 mg / kg administration group (FIG. 11 (d)).
- 2P10F 2-competitive ELISA For the purpose of epitope analysis of monkey cross-linking rat CD147 antibody, binding to 2P10F2ch IgG4P to CD147 recombinant protein was inhibited or examined by competitive ELISA.
- Human CD147-Fc fusion protein (Sino Biological Inc., 10186-H02H) was dissolved in PBS, adjusted to 20 ⁇ g / ml, 50 ⁇ l added to a 96 well plate (Thermofisher, Cat. 43454) and stored at 4 ° C. The protein solution was removed, 300 ⁇ l of PBS containing 1% BSA was added, and warmed at room temperature for 1 hour.
- r # 84, r # 101 and r # 131 inhibited the binding of 2P10F2ch IgG4P by 90% or more, suggesting that the antibody recognition site is close to 2P10F2.
- r # 110 did not inhibit the binding of 2P10F2 rat antibody. It is considered that the antibody recognition site may not be able to inhibit the binding of 2P10F2 rat antibody because it is separated from 2P10F2 or weakly bound.
- Example 3 Cloning of monkey-crossing rat antibody and preparation of human chimeric antibody 3) -1 Cloning of cDNA encoding variable region of rat anti-CD147 antibody Determination of nucleotide sequence 3) -1-1 variable of rat_CD147_ # 84 antibody Determination of Nucleotide Sequence of cDNA Encoding Region The procedure was carried out in the same manner as Example 1) -11-2.
- the nucleotide sequence of the cDNA encoding the variable region of the light chain of rat_CD147_ # 84 antibody determined is shown in SEQ ID NO: 48, and the amino acid sequence is shown in SEQ ID NO: 49.
- the nucleotide sequence of the cDNA encoding the variable region of the heavy chain is shown in SEQ ID NO: 50 and the amino acid sequence is shown in SEQ ID NO: 51.
- the CDRL1, CDRL2 and CDRL3 of the light chain variable region of the rat_CD147_ # 84 antibody are shown in SEQ ID NOs: 52, 53 and 54, respectively.
- the CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the rat_CD147_ # 84 antibody are shown in SEQ ID NOs: 55, 56 and 57, respectively.
- the CDRL1, CDRL2 and CDRL3 of the light chain variable region of the rat_CD147_ # 101 antibody are shown in SEQ ID NOs: 62, 63 and 64, respectively.
- the CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the rat_CD147_ # 101 antibody are shown in SEQ ID NOs: 65, 66 and 67, respectively.
- the CDRL1, CDRL2 and CDRL3 of the light chain variable region of the rat_CD147_ # 110 antibody are shown in SEQ ID NOs: 72, 73 and 74, respectively.
- the CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the rat_CD147_ # 110 antibody are shown in SEQ ID NOs: 75, 76 and 77, respectively.
- the CDRL1, CDRL2 and CDRL3 of the light chain variable region of the rat_CD147_ # 131 antibody are shown in SEQ ID NOs: 82, 83 and 84, respectively.
- the CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the rat_CD147_ # 131 antibody are shown in SEQ ID NOs: 85, 86 and 87, respectively.
- Example 3 -2-1-2 Construction of Light Chain Expression Vector of Human Chimeric Rat_CD147_ # 84
- the cDNA encoding the variable region of rat_CD147_ # 84 obtained in Example 3) -1-1 is used as a template, Example 1)
- a light chain expression vector of human chimeric rat_CD147_ # 84 was constructed in the same manner as in Example 12-12-4.
- the nucleotide sequence of the light chain of human chimeric rat_CD147_ # 84 and the amino acid sequence of the light chain are shown in SEQ ID NO: 89 and SEQ ID NO: 90, respectively.
- IgG1 type heavy chain expression vector of human chimeric rat_CD147_ # 84 The cDNA encoding the variable region of heavy chain of rat_CD147_ # 84 obtained in Example 3) -1-1 is used as a template
- an IgG1 type heavy chain expression vector of human chimeric rat_CD147_ # 84 was constructed in the same manner as in Example 1) -12-5.
- the nucleotide sequence of the IgG1 type heavy chain of human chimeric rat_CD147_ # 84 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 91 and SEQ ID NO: 92, respectively.
- IgG2 type heavy chain expression vector of human chimeric rat_CD147_ # 84 The cDNA encoding the variable region of heavy chain of rat_CD147_ # 84 obtained in Example 3) -1-1 is used as a template Then, an IgG2 type heavy chain expression vector of human chimeric rat_CD147_ # 84 was constructed in the same manner as in Example 1) -12-6. The nucleotide sequence of the IgG2 type heavy chain of human chimeric rat_CD147_ # 84 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively.
- IgG4P-type heavy chain expression vector of human chimeric rat_CD147_ # 84 The cDNA encoding the variable region of heavy chain of rat_CD147_ # 84 obtained in Example 3) -1-1 is used as a template Then, an IgG4 P-type heavy chain expression vector of human chimeric rat_CD147_ # 84 was constructed in the same manner as in Example 1) -13-6.
- the nucleotide sequence of the IgG4 P-type heavy chain of human chimeric rat_CD147_ # 84 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 95 and SEQ ID NO: 96, respectively.
- IgG1LALA-type heavy chain expression vector of human chimeric rat_CD147_ # 84 The cDNA encoding the variable region of heavy chain of rat_CD147_ # 84 obtained in Example 3) -1-1 is used as a template Then, an IgG1 LALA type heavy chain expression vector of human chimeric rat_CD147_ # 84 was constructed in the same manner as in Example 1) -13-4.
- the nucleotide sequence of the IgG1 LALA type heavy chain of human chimeric rat_CD147_ # 84 and the amino acid sequence of said heavy chain are shown in SEQ ID NO: 97 and SEQ ID NO: 98, respectively.
- an IgG4 PFALA type heavy chain expression vector of human chimeric rat_CD147_ # 84 by inserting the amplified DNA fragment using the In-Fusion HD PCR cloning kit (Clontech) at the site where pCMA-G4 PFALA was digested with restriction enzyme BlpI. did.
- the nucleotide sequence of the IgG4 PFALA type heavy chain of human chimeric rat_CD147_ # 84 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 99 and SEQ ID NO: 100, respectively.
- IgG2 type heavy chain expression vector of human chimeric rat_CD147_ # 101 The cDNA encoding the variable region of heavy chain of rat_CD147_ # 101 obtained in Example 3) -1-2 is used as a template
- an IgG2 type heavy chain expression vector of human chimeric rat_CD147_ # 101 was constructed in the same manner as in Example 1) -12-6.
- the nucleotide sequence of the IgG2 type heavy chain of human chimeric rat_CD147_ # 101 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 103 and SEQ ID NO: 104, respectively.
- IgG4P-type heavy chain expression vector of human chimeric rat_CD147_ # 101 The cDNA encoding the variable region of heavy chain of rat_CD147_ # 101 obtained in Example 3) -1-2 is used as a template Then, an IgG4 P-type heavy chain expression vector of human chimeric rat_CD147_ # 101 was constructed in the same manner as in Example 1) -13-6.
- the nucleotide sequence of the IgG4 P-type heavy chain of human chimeric rat_CD147_ # 101 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 105 and SEQ ID NO: 106, respectively.
- IgG4PFALA type heavy chain expression vector of human chimeric rat_CD147_ # 101 The cDNA encoding the variable region of heavy chain of rat_CD147_ # 101 obtained in Example 3) -1-2 is used as a template Then, an IgG4 PFALA type heavy chain expression vector of human chimeric rat_CD147_ # 101 was constructed in the same manner as in Example 3) 2- 1-7.
- the nucleotide sequence of the IgG4 PFALA type heavy chain of human chimeric rat_CD147_ # 101 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 107 and SEQ ID NO: 108, respectively.
- IgG2 type heavy chain expression vector of human chimeric rat_CD147_ # 110 The cDNA encoding the variable region of the heavy chain of rat_CD147_ # 110 obtained in Example 3) -1-3 is used as a template
- an IgG2-type heavy chain expression vector of human chimeric rat_CD147_ # 110 was constructed in the same manner as in Example 1) 12-6.
- the nucleotide sequence of the IgG2 type heavy chain of human chimeric rat_CD147_ # 110 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 111 and SEQ ID NO: 112, respectively.
- IgG4 P-type heavy chain expression vector of human chimeric rat_CD147_ # 110 The cDNA encoding the variable region of the heavy chain of rat_CD147_ # 110 obtained in Example 3) -1-3 is used as a template Then, an IgG4 P-type heavy chain expression vector of human chimeric rat_CD147_ # 110 was constructed in the same manner as in Example 1) -13-6.
- the nucleotide sequence of the IgG4 P-type heavy chain of human chimeric rat_CD147_ # 110 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 113 and SEQ ID NO: 114, respectively.
- IgG4PFALA type heavy chain expression vector of human chimeric rat_CD147_ # 110 The cDNA encoding the variable region of heavy chain of rat_CD147_ # 110 obtained in Example 3) -1-3 is used as a template Then, an IgG4 PFALA type heavy chain expression vector of human chimeric rat_CD147_ # 110 was constructed in the same manner as in Example 3) 2- 1-7.
- the nucleotide sequence of the IgG4 PFALA type heavy chain of human chimeric rat_CD147_ # 110 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 115 and SEQ ID NO: 116, respectively.
- Example 3 an IgG2 type heavy chain expression vector of human chimeric rat_CD147_ # 131 was constructed in the same manner as in Example 1) 12-6.
- the nucleotide sequence of the IgG2 type heavy chain of human chimeric rat_CD147_ # 131 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 119 and SEQ ID NO: 120, respectively.
- IgG4P-type heavy chain expression vector of human chimeric rat_CD147_ # 131 The cDNA encoding the variable region of the heavy chain of rat_CD147_ # 131 obtained in Example 3) -1-4 is used as a template Then, an IgG4 P-type heavy chain expression vector of human chimeric rat_CD147_ # 131 was constructed in the same manner as in Example 1) -13-6.
- the nucleotide sequence of the IgG4 P-type heavy chain of human chimeric rat_CD147_ # 131 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 121 and SEQ ID NO: 122, respectively.
- the human chimeric antibody of rat_CD147_ # 84 obtained by combining the IgG1 type heavy chain expression vector of human chimeric rat_CD147_ # 84 and the light chain expression vector of human chimeric rat_CD147_ # 84 was named “# 84 chIgG1”.
- the human chimeric antibody of rat_CD147_ # 84 obtained by combining the IgG2 type heavy chain expression vector of human chimeric rat_CD147_ # 84 and the light chain expression vector of human chimeric rat_CD147_ # 84 was named “# 84 chIgG2”.
- a human chimeric antibody of rat_CD147_ # 84 obtained by combining an IgG4P-type heavy chain expression vector of human chimeric rat_CD147_ # 84 and a light chain expression vector of human chimeric rat_CD147_ # 84 was named “# 84 chIgG4P”.
- a human chimeric antibody of rat_CD147_ # 84 obtained by combining an IgG1 LALA type heavy chain expression vector of human chimeric rat_CD147_ # 84 and a light chain expression vector of human chimeric rat_CD147_ # 84 was named “# 84 chIgG1 LALA”.
- a human chimeric antibody of rat_CD147_ # 84 obtained by combining an IgG4 PFALA type heavy chain expression vector of human chimeric rat_CD147_ # 84 and a light chain expression vector of human chimeric rat_CD147_ # 84 was named “# 84 chIgG4 PFALA”.
- the human chimeric antibody of rat_CD147_ # 101 obtained by combining the IgG2 type heavy chain expression vector of human chimeric rat_CD147_ # 101 and the light chain expression vector of human chimeric rat_CD147_ # 101 was named “# 101 chIgG2”.
- a human chimeric antibody of rat_CD147_ # 101 obtained by combining an IgG4P type heavy chain expression vector of human chimeric rat_CD147_ # 101 and a light chain expression vector of human chimeric rat_CD147_ # 101 was named “# 101 chIgG4P”.
- a human chimeric antibody of rat_CD147_ # 101 obtained by combining an IgG4 PFALA type heavy chain expression vector of human chimeric rat_CD147_ # 101 and a light chain expression vector of human chimeric rat_CD147_ # 101 was named “# 101 chIgG4 PFALA”.
- the human chimeric antibody of rat_CD147_ # 110 obtained by combining the IgG2 type heavy chain expression vector of human chimeric rat_CD147_ # 110 and the light chain expression vector of human chimeric rat_CD147_ # 110 was named “# 110 chIgG2”.
- a human chimeric antibody of rat_CD147_ # 110 obtained by combining an IgG4P-type heavy chain expression vector of human chimeric rat_CD147_ # 110 and a light chain expression vector of human chimeric rat_CD147_ # 110 was named “# 110 chIgG4P”.
- a human chimeric antibody of rat_CD147_ # 110 obtained by combining an IgG4 PFALA type heavy chain expression vector of human chimeric rat_CD147_ # 110 and a light chain expression vector of human chimeric rat_CD147_ # 110 was named “# 110 chIgG4 PFALA”.
- the human chimeric antibody of rat_CD147_ # 131 obtained by combining the IgG2 type heavy chain expression vector of human chimeric rat_CD147_ # 131 and the light chain expression vector of human chimeric rat_CD147_ # 131 was named “# 131 chIgG2”.
- a human chimeric antibody of rat_CD147_ # 131 obtained by combining an IgG4P-type heavy chain expression vector of human chimeric rat_CD147_ # 131 and a light chain expression vector of human chimeric rat_CD147_ # 131 was named “# 131 chIgG4P”.
- Example 3 Purification of Human Chimeric Antibody of Monkey Cross-Positive Rat Antibody From the culture supernatant obtained in Example 3) -3-1, it was purified by the same method as in Example 1) -14-2.
- Example 4 In Vivo Antitumor Activity of Human Chimeric Antibody Human pancreatic strain MIA PaCa-2 of 5 ⁇ 10 6 cells was suspended in PBS containing 50% GFR-Matrigel (Corning, Cat. 354230), and 5-6 The axillary region of a female NOD-scid mouse (NOD. CB17-Prkdc ⁇ scid> / J, purchased from Charles River Japan) was implanted subcutaneously in the axillary region of a female.
- NOD. CB17-Prkdc ⁇ scid> / J purchased from Charles River Japan
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- Human chimeric antibody # 84 showed a dose-dependent anti-tumor effect for all IgG subtypes.
- # 84 ch IgG4P 10 mg / kg administration group in which the strongest antitumor effect was confirmed, complete regression of the tumor was confirmed in 5 mice out of 5 after 20 days of transplantation.
- Human chimeric antibody # 101 showed a dose-dependent anti-tumor effect for all IgG subtypes.
- IgG4P 10 mg / kg administration group in which the strongest antitumor effect was confirmed complete regression of the tumor was confirmed in 4 mice out of 5 20 days after transplantation.
- Human chimeric antibody # 110 exhibited a dose-dependent anti-tumor effect for all IgG subtypes. In the 110 ch IgG 4 P 10 mg / kg administration group in which the strongest antitumor effect was confirmed, complete regression of the tumor was confirmed in 3 mice out of 5 22 days after transplantation.
- Human chimeric antibody # 131 showed a dose-dependent anti-tumor effect at the IgG4P subtype. In the case of # 131 ch IgG2, there was no difference in the antitumor effect observed in the 3 mg / kg and 10 mg / kg administration groups.
- Example 5 CD147 Binding Evaluation of Human Chimeric Antibody
- Example 3 Measurement of dissociation constant of # 84 chIgG1, # 84 chIgG2, # 84 chIgG4P, # 84 chIgG1 LALA, # 84 chIgG4 PFALA, # 101 chIgG4P, # 110 chIgG4P against human CD147 prepared in -3-1
- the antibody is captured (captured) as a ligand on an Anti-Human IgG (Fc) antibody immobilized using Human Antibody Capture Kit (manufactured by GE Healthcare Bioscience) using Biacore T200 (manufactured by GE Healthcare Bioscience).
- HBS-EP + manufactured by GE Healthcare Bioscience
- CM5 manufactured by GE Healthcare Bioscience
- a dilution series solution of CD147 protein as an antigen (0.25-4 ⁇ g / mL for # 131 ch IgG4 P, # 84 ch IgG1, #) 84 ch IgG2, # 84 ch IgG 4 P, # 84 ch IgG 1 LALA, # 84 ch IgG 4 PFALA, # 101 ch IgG 4 P, 0.5 to 8 ⁇ g / mL against # 110 ch IgG 4 P) is added at a flow rate of 30 ⁇ L / min for 120 seconds, followed by a dissociation phase of 120 seconds, 300 seconds or 600 seconds Was monitored.
- the CD147 protein was expressed in E. coli and purified after cleaving and purifying the tag after 2 steps purification of Ni affinity, SEC.
- 3 M magnesium chloride manufactured by GE Healthcare Bioscience was added for 30 seconds at a flow rate of 20 ⁇ L / min.
- rat_CD147_ # 84 The framework region of rat_CD147_ # 84 is described by KABAT et al. Consensus sequences of human kappa chain subgroup 1 and human gamma chain subgroup 3 as defined in (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)) Because of their high homology to the sequences, they were respectively selected as acceptors for the light and heavy chains of rat_CD147_ # 84.
- rat_CD147_ # 101 The framework region of rat_CD147_ # 101 is described by KABAT et al. Consensus sequences of human kappa chain subgroup 1 as defined in and IGHV3-30 * 05 and IGHJ3 * of human gamma chain defined in human gamma chain subgroup 3 and IMGT (THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM®). Since they have high homology to 01, they were respectively selected as light chain and heavy chain acceptors of rat_CD147_ # 101.
- the framework region of rat_CD147_ # 110 has high homology to IGKV1-39 * 01 and IGKJ4 * 01 of human kappa chain and IGHV1-2 * 02 and IGHJ6 * 01 of human gamma chain defined in IMGT. From, they were selected as light and heavy chain acceptors of rat_CD147_ # 110 respectively.
- the framework region of rat_CD147_ # 131 has high homology to IGKV1-39 * 01 and IGKJ2 * 01 of human kappa chain and IGHV3-30 * 05 and IGHJ6 * 01 of human gamma chain defined in IMGT. And were selected as the light chain and heavy chain acceptors of rat_CD147_ # 131 respectively. Donor residues to be transferred onto the acceptor are described by Queen et al. The three-dimensional model was selected by analysis with reference to the criteria given by (Proc. Natl. Acad. Sci. USA 86, 10029-1 0033 (1989)).
- Humanized antibody heavy chains in which human IgG2 and IgG4P gamma chain constant regions were connected to the designed variable regions were designed, and named # 84H1hIgG2 and # 84H1hIgG4P, respectively.
- the full-length amino acid sequence of # 84H1 hIgG2 is set forth in SEQ ID NO: 123.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 123 is set forth in SEQ ID NO: 124.
- the full-length amino acid sequence of # 84H1 hIgG4P is set forth in SEQ ID NO: 125.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 125 is set forth in SEQ ID NO: 126.
- a humanized antibody light chain was designed, in which a human ⁇ ⁇ chain constant region was connected to the designed variable region, and it was named # 84L2h.
- the full-length light chain amino acid sequence of # 84L2h is set forth in SEQ ID NO: 127.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 127 is set forth in SEQ ID NO: 128.
- Humanized antibody heavy chain # 101H1h1 by transferring the amino acid residue of the acceptor shown in FIG. 15A to the variable region of rat_CD147_ # 101 heavy chain: The variable area of was designed.
- Humanized antibody heavy chains in which human IgG2 and IgG4P gamma chain constant regions were connected to the designed variable regions were designed, and named # 101H1hIgG2 and # 101H1hIgG4P, respectively.
- the full-length amino acid sequence of # 101H1 hIgG2 is set forth in SEQ ID NO: 129.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 129 is set forth in SEQ ID NO: 130.
- the full-length amino acid sequence of # 101H1 hIgG4P is set forth in SEQ ID NO: 131.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 131 is set forth in SEQ ID NO: 132.
- Humanized antibody heavy chains in which the constant chain region of IgG4P was connected to the designed variable region were designed and named # 110 H1 h IgG4 P and # 110 H13 h IgG4 P, respectively.
- the full-length amino acid sequence of # 110H1 hIgG4P is shown in SEQ ID NO: 135, and the full-length amino acid sequence of # 110 H13 hIgG4P is shown in SEQ ID NO: 147.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 135 is set forth in SEQ ID NO: 136
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 147 is set forth in SEQ ID NO: 148.
- variable regions of humanized antibody light chain # 110L4h, # 110L2h and # 110L12h were designed.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 137 is described in SEQ ID NO: 138, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 149 in SEQ ID NO: 150, and the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 151 in SEQ ID NO: 152 .
- a humanized antibody heavy chain in which a human IgG2 gamma chain constant region was connected to the designed variable region was designed and designated # 131H2hIgG2.
- the full-length amino acid sequence of # 131H2h IgG2 is set forth in SEQ ID NO: 139.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 139 is set forth in SEQ ID NO: 140.
- a humanized antibody light chain was designed, in which the constant region of human ⁇ chain was connected to the designed variable region, and it was named # 131L2h.
- the full-length light chain amino acid sequence of # 131L2h is set forth in SEQ ID NO: 141.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 141 is set forth in SEQ ID NO: 142.
- Example 6 -2-2 Construction of # 101L2h Expression Vector The DNA fragment shown in nucleotide numbers 37 to 402 of the nucleotide sequence of # 101L2h shown in SEQ ID NO: 134 was synthesized (GENEART).
- Example 6) A # 101L2h expression vector was constructed in the same manner as in Example 2-1.
- # 84H1 hIgG4P Expression Vector The DNA fragment shown in nucleotide numbers 36 to 437 of the nucleotide sequence of # 84H1 h IgG4 P shown in SEQ ID NO: 126 was synthesized (GENEART). Using the In-Fusion HD PCR cloning kit (Clontech), the # 84H1hIgG4P expression vector was constructed by inserting the synthesized DNA fragment into the site where pCMA-G4P was digested with restriction enzyme BlpI.
- # 110H1hIgG4P and # 110H13hIgG4P Expression Vector The DNA fragment shown in nucleotide numbers 36 to 425 of the nucleotide sequences of # 110H1hIgG4P and # 110H13hIgG4P shown in SEQ ID NO: 136 and SEQ ID NO: 148 was synthesized (GENEART) .
- # 110H1 hIgG4P and # 110H13 hIgG4P expression vectors were constructed in the same manner as in 3-2.
- Example 6 -4 Preparation of Humanized Antibody 6) -4-1 Production of Humanized Antibody
- the antibody was produced in the same manner as in Example 1) -14-1. H corresponding to the combination of H chain and L chain shown in Example 6) -1-5, Example 6) -1-8, Example 6) -1-11, and Example 6) -1-14 Various humanized antibodies were obtained by combining the chain expression vector and the L chain expression vector.
- Example 6 -4-2 One-Step Purification of Humanized Antibody
- the antibody was purified from the culture supernatant obtained in Example 6) -4-1 by a one-step process of rProtein A affinity chromatography.
- the culture supernatant was applied to a column (manufactured by GE Healthcare Bioscience) packed with PBS equilibrated with MabSelectSuRe, and then the column was washed with 2 column volumes or more of PBS. Then, it was eluted with 2 M arginine hydrochloride solution (pH 4.0), and the fractions containing the antibody were collected.
- the fraction was subjected to buffer substitution into PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette).
- the antibody was concentrated with Centrifugal UF Filter Device VIVASPIN 20 (fractional molecular weight UF10K, Sartorius) to adjust the IgG concentration to 1 mg / mL or more. Finally, the resultant was filtered with Minisart-Plus filter (Sartorius) to obtain a purified sample.
- VIVASPIN 20 fractional molecular weight UF10K, Sartorius
- Example 6 Two-Step Purification of Humanized Antibody
- the culture supernatant obtained in Example 6) -4-1 was purified by a two-step process of rProtein A affinity chromatography and ceramic hydroxyapatite.
- the culture supernatant was applied to a column (manufactured by GE Healthcare Bioscience) packed with PBS equilibrated MabSelectSuRe, and then the column was washed with 2 column volumes or more of PBS.
- the antibody was then eluted with 2 M arginine hydrochloride solution (pH 4.0).
- the fractions containing the antibody were subjected to buffer substitution into PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) and diluted 5-fold with a buffer of 5 mM sodium phosphate / 50 mM MES / pH 7.0 Later, it was applied to a ceramic hydroxyapatite column (Bio-Scale CHT Type-1 Hydroxyapatite Column, Japan Bio-Rad) equilibrated with a buffer of 5 mM NaPi / 50 mM MES / 30 mM NaCl / pH 7.0. Linear gradient elution with sodium chloride was performed to collect fractions containing the antibody.
- the fraction was subjected to buffer substitution to HBSor (25 mM histidine / 5% sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette).
- the antibody was concentrated with Centrifugal UF Filter Device VIVASPIN 20 (fractional molecular weight UF10K, Sartorius) to adjust the IgG concentration to 25 mg / mL.
- the resultant was filtered with Minisart-Plus filter (Sartorius) to obtain a purified sample.
- Example 7 Activity measurement of humanized antibody 7) -1 CD147 antibody binding evaluation of humanized antibody
- Example 6) Humanized anti-human CD147 antibody # 84H1L2hIgG2, # 84H1L2hIgG4P, # 101H1L2hIgG2, prepared in -4-3 The dissociation constant of # 101H1L2hIgG4P, # 110H1L4hIgG4P and # 131H2L2hIgG2 and human CD147 was measured using Biacore T200 (manufactured by GE Healthcare Bioscience) and using Anti Antibody immobilized on Human Antibody Capture Kit (manufactured by GE Healthcare Bioscience).
- the antibody is captured (captured) as a ligand in Human IgG (Fc) antibody, and the antigen is measured as an analyte. It was carried out by capture method that. HBS-EP + (manufactured by GE Healthcare Bioscience) was used as a running buffer, and CM5 (manufactured by GE Healthcare Bioscience) was used as a sensor chip.
- a dissociation time of 300 seconds was subsequently monitored by adding # # 4 4H2LIgG2, # 84H1L2hIgG4P, # 1 10H1L4hIgG4P, # 1 10H13L2h IgG4P, # 1 10H13L12hIgG4P, # 110H13L12hIgG4P and # 131H2L2hIgG2 (0.25 to 4 ⁇ g / mL) at a flow rate of 30 ⁇ L / min for 120 seconds, and subsequently for 300 seconds.
- gemcitabine purchased from Eli Lilly Japan
- the major axis and minor axis of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated by the following formula.
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- the tumor growth inhibition rate of the control drug is 71%, no disappearance of the tumor was observed, whereas any humanized CD147 antibody was superior to gemcitabine in the 3 or 10 mg / kg dose group. It showed a tumor effect.
- the 10 mg / kg administration group of # 84H1L2hIg4P, # 101H1L2hIg2, # 101H1L2hIg4P, and # 110H1L4hIg4P a strong antitumor effect accompanied by disappearance of tumor was confirmed.
- Example 8 Activation of in vitro p38 MAPK signal of CD147 antibody 8)-Induction of p38 MAPK phosphorylation by anti-human CD147 human chimeric antibody in pancreatic cancer cell PANC-1-CD147 promotes phosphorylation of p38 MAPK by activation (Lim et al., FEBS Letters, 88-92, 1998) (Li et., Al. J. Hepatology, 1378-1389, 2015).
- Simple Western assays method for p38 MAPK phosphorylation protein The evaluation was made using Simple Japan Co., Ltd., Wes.
- PANC-1 cells were treated similarly with 10 ⁇ g / ml of human IgG (Jackson ImmunoResearch, 009-000-003) and subjected to analysis.
- p38 MAPK p38 MAPK rabbit mAb (Cell signaling technology, Cat. # 9212) was used.
- P-p38 MAPK T180 / Y182) (D3F9)
- XP rabbit mAb (Cell signaling technology, # 4511 S) was used.
- the ratio of phosphorylated p38 MAPK signal to detected p38 MAPK signal is shown in FIG. Antibody-treated samples were run in duplicate and the mean values are shown in the figure.
- the ratio of Ser 82 or Ser 15 phosphorylated HSP 27 signal to detected HSP 27 signal is shown in FIG. 20 (a) or FIG. 20 (b), respectively.
- Antibody-treated samples were run in duplicate and the mean values are shown in the figure.
- Example 9 Intratumoral p38 signal activation of CD147 antibody Does the activation of p38 MAPK signal by the CD147 antibody observed in vitro also occur in the tumor formed in mouse subcutaneous tissue? MIA PaCa-2 mouse subcutaneous tumor I checked it out. A 5 ⁇ 10 6 human pancreatic strain MIA PaCa-2 is suspended in PBS containing 50% GFR-Matrigel (Corning, Cat. 354230), and 5-week-old female NOD-scid mice (NOD. CB17-Prkdc ⁇ scid> / J (purchased from Japan Charles River).
- Grouping was performed 5 days after transplantation based on tumor volume, and anti-human CD147 human chimeric antibody LN22R8chIgG4P was administered intraperitoneally to tumor-bearing mice at 10 mg / kg on the day after grouping.
- Tumors were collected at 6, 24, 48, 72 hours after antibody administration, frozen with dry ice, and stored frozen.
- the prepared tumor lysate was analyzed using the Simple Western assays method (Protein Simple Japan Co., Ltd., Wes).
- the average value of the detected p38 MAPK signal is shown in FIG. 21 (a) together with the standard error.
- the average value of the detected phosphorylated p38 MAPK signal is shown in FIG. 21 (b) together with the standard error.
- the average value of the ratio of phosphorylated p38 MAPK signal to p38 MAPK signal is shown in FIG. 21 (c) together with the standard error.
- LN22R8 chIgG4P increased the proportion of phosphorylated P38 MAPK from 6 hours to 72 hours after administration. From 24 hours to 72 hours, the signal of phosphorylated p38 MAPK decreased to a peak for 6 hours due to the increase of p38 MAPK.
- Example 10 Induction of molecule CXCL8 downstream of p38 MAPK Activation of p38 MAPK induces induction of CXCL8 via mRNA stabilization (Hoffmann et al., J. Leukoc. Biol., 847-855, 2002), SMAD3 / 4. Signal activation has been reported (Leovonen et al., PLOS ONE, e57474, 2013).
- MIA PaCa-2 tumors RNA extracted from mouse subcutaneous tumors in which CXCL8 gene expression was induced after antibody administration or after antibody administration was examined by quantitative PCR.
- LN22R8chIgG1, LN22R8chIgG2 or LN22R8chIgG4P prepared in Example 1) -14 was used.
- EXPRESS One-Step SuperScript qRT-PCR kit Universal Thermofisher scientific, Cat. 11781-01K
- ipo8 Thermo Fisher Scientific, Cat. Hs00183533_m1
- tbp TATA box binding protein
- rho B rashomolog family member B
- Example 11 Induction of cxcl8, rhoB gene in enhancement of anti-tumor effect by human chimeric antibodyification of anti-human CD147 rat antibody
- the anti-human CD147 rat antibody rat_CD147_ # 110 is as shown in Example 2, Example 7, Anti-tumor effect was enhanced by human chimerization.
- Rat_CD147_ # 110 and chimeric antibody # 110 chIgG4P were compared in the same manner as 14) -2 for the induction of cxcl8 and rhoB observed in tumors after administration of human chimeric antibodies LN22R8 chIgG1, LN22 R8 chIgG2 or LN22 R8 chIgG4P.
- SMAD4 pancreatic cancer tumor-bearing model
- a transcription factor, SMAD4 is known as one of the important molecules for activation of SMAD signal (Zang, et al., Current Biology, 270-276. , 1997).
- SMAD signal is partially inhibited due to genetic deficiency of SMAD 4 (Hahn, et al., Science, 350-353, 1996).
- Anti-tumor effect of CD147 antibody on pancreatic cancer cell line BxPC-3 deficient in SMAD4 and partially blocked by SMAD signal (Yasutome et al., Clin. Exp. Metastasis, 461-473, 2005) I checked.
- the major axis and minor axis of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated by the following formula.
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm) It showed in FIG. The graph shows the mean value and the standard error together with the change of the tumor volume.
- BxPC-3 tumors were resistant to LN22R8 antibody, 2P10F2 antibody and gemcitabine.
- Example 13 Acquisition of CD147 Antibody Susceptibility by Introducing SMAD4 It has been reported that the SMAD signal is restored by introducing SMAD4 in multiple pancreatic cancer cell lines. It was examined whether the sensitivity of the CD147 antibody was increased by the SMAD signal recovered by the introduction of SMAD4.
- Retro-X TM Q vector kit Retroviral vector (Takara Bio Inc., Retro-X TM Q Vector Set , Cat.631516) introducing the human SMAD4 gene prepared by artificial synthesis cloning site of pQCXIP included in the kit as to obtain a SMAD4 expression retroviral vector.
- Retro-X Universal Packaging System Takara Bio Inc., Cat. 631530
- SMAD4 expression retrovirus vector is introduced into BxPC-3, and virus infection with puromycin (Takara Bio Inc., Cat.
- BxPC-3 cell which became SMAD4 positive was selected, and it was referred to as SMAD4 positive BxPC-3 cell, BxPC-3-SMAD4.
- the retrovirus vector pQCXIP was similarly infected, and BxPC-3 cells which became puromycin resistant were designated as BxPC-3-mock. Retrovirus infection experiments were performed twice, and for BxPC-3-mock, BxPC-3-SMAD4, lot. 1, lot. 2 was produced.
- MIA PaCa-2 was SMAD4 positive and CD147 positive.
- BxPC-3 was SMAD4 negative and CD147 positive.
- BxPC-3-mock was SMAD4 negative and CD147 positive without any influence of retrovirus infection.
- BxPC-3-SMAD4 into which the SMAD4 expression retroviral vector was introduced, was used for the lot. 1, lot. All of 2 were SMAD4 positive. Lot. SMAD4 expression of 2 is high, and in later experiments, lot. 2 was used as BxPC-3-SMAD4.
- BxPC-3-SMAD4 100% Matrigel (Corning, Cat. 354234) And suspended in the axillary region of 5-week-old female BALB / c-nu mice (CAnN.Cg-Foxn1nu / CrlCrlj, purchased from Japan Charles River).
- BxPC-3-SMAD4 was divided into groups three days after transplantation, respectively, and human chimera on days 7, 14, 21 and 28 days after classification.
- the major axis and minor axis of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated by the following formula.
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in FIG. 25 (b) and FIG. 25 (c).
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- BxPC-3-mock tumors were resistant or insensitive to LN22R8R8chIgG2 or LN22R8R8chIgG4P antibodies.
- BxPC-3-SMAD4 tumors showed partial sensitivity to LN22R8R8chIgG2 or LN22R8R8chIgG4P antibodies.
- Example 1 In the same manner as in the section-18), LNA22R8chIgG4P was administered as an anti-human CD147 human chimeric antibody to a mouse on which 10 mg / kg of MIA PaCa-2 subcutaneous tumor was engrafted.
- gentleMACS Octo Dissociator with Heaters (Milteny) was used.
- FIG. 26 (a) is a graph showing the average value and standard error of the measurement values of three tumor samples for the p38 MAPK measurement value.
- FIG. 26 (b) is a graph showing the mean values and standard errors of the measured values of the three tumor samples for the phosphorylated p38 MAPK measured values.
- p38 and phosphorylated p38 were increased more than 2-fold.
- a partial decrease of p38 and a partial increase of phosphorylated p38 were observed 72 hours after administration of the CD147 human chimeric antibody.
- SMAD4 results in the enhancement of p38 signal. It is possible that the increased sensitivity of the SMAD4-dependent p38 signal may contribute to the increased sensitivity of the CD147 human chimeric antibody LN22R8 chIgG4P.
- Example 14 Antitumor Effect of Chimeric CD147 Antibody in Gemcitabine-Resistant Pancreatic Cancer The antitumor effect of CD147 antibody in a gemcitabine-resistant pancreatic cancer tumor model was examined.
- a 5 ⁇ 10 6 human pancreatic strain MIA PaCa-2 is suspended in PBS containing 50% GFR-Matrigel (Corning, Cat. 354230), and 5-week-old female NOD-scid mice (NOD. CB17-Prkdc ⁇ scid> / J, implanted in the axillary region of Japan Charles River).
- grouping day On day 20 (grouping day), on day 20, 10 mg / kg was intraperitoneally administered.
- the major axis and minor axis of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated by the following formula.
- the mean size of the tumors in the group to which the control drug gemcitabine was administered was 1269 mm 3 28 days after transplantation, and no regression was observed, whereas the mean size of the tumors in the group coadministered with the CD147 human chimeric antibody LN22R8chIgG4P Is 15 mm 3 and regression of tumor was observed in 3 out of 5 cases. From these results, it has been shown that the pancreatic cancer tumor which is resistant to gemcitabine and proliferates may also be sensitive to the CD147 antibody.
- Example 15 Antitumor effect of humanized CD147 antibody in liver cancer model 15) -1 Expression of CD147 and SMAD4 in Hep G2 cells Similar to Example 13) -2, liver cancer cell line HepG2 cells (ATCC, Cat) The expression of CD147 and SMAD4 was confirmed.
- MIA PaCa-2, BxPC-3 As a control sample of SMAD4 positive, BxPC-3 was used as a control sample of MIA PaCa-2 and SMAD4 negative, respectively.
- the signal ratio to GAPDH was determined and summarized below. Hep G2 cells were found to be positive for CD147, SMAD4.
- Hep G2 cells treated with MEM-M6 / 1-APC showed a strong fluorescence signal and were found to express CD147.
- an anti-human CD147 human chimeric antibody (LN22R8chIgG4P) or Example 6) HepG2 cells (ATCC, CatHB) treated with 10 ⁇ g / ml of anti-human CD147 humanized antibody (# 84H1L2hIgG2, # 84H1L2hIg4P, # 101H1L2hIgG2, # 101H1L2hIgG4P, # 110H1L4hIg4P, # 131H2L2hIgG2) prepared in -4-2 for 15 minutes.
- P80 phosphorylation was evaluated using the Simple Western assays method (Protein Simple Japan Co., Ltd., Wes) for 8065).
- HepG2 cells were treated similarly with 10 ⁇ g / ml of human IgG (hIgG, Jackson ImmunoResearch, 009-000-003) and subjected to analysis.
- p38 MAPK rabbit mAb (Cell signaling technology, Cat. # 9212) was used.
- P-p38 MAPK T180 / Y182) (D3F9)
- XP rabbit mAb was used.
- the ratio of phosphorylated p38 MAPK signal to the detected p38 MAPK signal is shown in FIG. 28 (b).
- Treatment with LN22R8chIgG4P, # 84H1L2hIgG2, # 84H1L2hIg4P, # 101H1L2hIgG2, # 101H1L2hIgG4P, # 110H1L4hIg4P or # 131H2L2hIgG2 also increases phosphorylation by at least twice as much as that of human IgG-treated p38 MAPK Similarly, it was found that phosphorylation of p38 MAPK was induced by anti-human CD147 antibody.
- a 5 ⁇ 10 6 human liver strain HepG2 was suspended in PBS containing 50% Matrigel (Corning, Cat. 354234), and 4-week-old female NOD-scid mice (NOD. CB17-Prkdc ⁇ scid> / J, The graft was implanted subcutaneously in the axillary region of Japan Charles River). Grouping was performed 9 days after transplantation based on the tumor volume, and the humanized CD147 antibody (# 84H1L2hIgG2, # 84H1L2hIg4P, # 110H1L4hIg4P) prepared with human chimeric CD147 antibody (LN22R8chIgG4P), Example 6-4-2 was used.
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in Table 7 and FIGS. 29 (a) to (d).
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- LN22R8chIgG4P, # 84H1L2hIgG2, # 84H1L2hIg4P, and # 110H1L4hIg4P all exhibited excellent antitumor effects accompanied by the disappearance of tumors.
- CD147 Effects on T cells and PBMCs CD147 is up-regulated on CD4 positive and CD8 positive T cells as T cells are activated (Hu et al., J. Cell. Mol Med., 2132-2143) , 2010), some CD147 antibodies have been reported to have the effect of inhibiting T cell activation-inducing ability and proliferation (Koch et al., Int. Immunology, 777-786, 1999; Chiampanichayakul et al., Immunology 167-178, 2006).
- PBL peripheral blood lymphocytes
- an APC-labeled anti-human CD147 mouse IgG1 antibody MEM-M6 / 1-APC (CD147-APC, Thermofisher, Cat. MA1-10104) was used as a commercially available anti-human CD147 antibody.
- mIgG1-APC Milteny Bio Inc., Cat. 130-092-214 was used.
- APC / Fire TM 750 anti -human CD3 Antibody (BioLegend Co., Cat.344840), PerCP / Cy5.5 anti -human CD4 (BioLegend , Cat. 344 608), Brilliant Violet 510 anti-human CD8 (BioLegend, Cat. 343732) was used.
- CD147-APC The binding of CD147-APC in CD3, CD4 positive cells and CD3, CD8 positive cells is summarized in FIG.
- CD3 / CD28 beads are stimulated in CD3 and CD4 positive cells and CD3 and CD8 positive cells, increased expression of CD147 is confirmed, and the expression of CD147 on the surface of T cell membrane is increased with T cell activation. That was confirmed.
- cytokines IL2, TNF ⁇ , INF ⁇
- IL2 Quantikine ELISA Human IL-2 (R & D systems, Cat. D2050) was used.
- TNF ⁇ Amersham TNF- ⁇ Human, Biotrak Easy ELISA (GE Healthcare Japan Ltd., Cat. RPN 5967) was used.
- Human IFN- ⁇ ELISA development kit (MABTECH, Cat. 3420-1H-6) was used for measurement of IFN ⁇ . The measurement was performed in triplicate, and the average value and standard deviation of the detected absorbance were calculated and summarized in FIG.
- Cytokine (IL2, TNF ⁇ , INF ⁇ ) produced from the measured PBL were all confirmed to rise only in the wells to which the positive control CD3 / CD28-beads had been added, and any anti-human CD147 human chimeric antibody coat Even in the well culture, no increase in cytokine was observed as in the negative control hIgG.
- Example 17 X-ray crystallographic analysis of a humanized # 110 Fab'-CD147 complex 17) -1 Crystallization of a complex: Dithiothreitol reduces Fab'2 obtained by cleaving humanized # 110 H1 L4 h IgG4 P with Pepsin Then, they were alkylated with Iodoacetamide to obtain Fab 'fragments. The mixture of this Fab ′ fragment and hCD147v2 (22-205) used in Example 5) was donated to gel filtration chromatography using a Superdex 10/300 GL Increase column (GE Healthcare) to obtain a complex fraction.
- the complex was replaced with Amicon Ultra 15 MWCO 10K (manufactured by Millipore) in buffer (10 mM Tris HCl pH 7.5, 50 mM NaCl) and concentrated to 13 g / L.
- the complex solution was crystallized by vapor diffusion.
- a solution prepared by adding an equal amount of a precipitant solution (0.1 M NaMalonate pH 7.0, 12% (w / v) Polyethylene Glycol 3350) to 0.5 ⁇ L of a protein solution is sealed with 0.05 mL of the precipitant solution
- the container was stored so that both solutions did not touch, and was left at 25 ° C.
- the 0.15 mm x 0.15 mm x 0.3 mm crystals obtained after about one week were immersed in a precipitant solution to which Polyethylene Glycol 400 was added to be 30% (w / v) and then frozen with liquid nitrogen .
- X-ray diffraction data were collected at beamline PF-BL17A of the synchrotron radiation facility Photon Factory (Ibaraki Prefecture, Japan). From the obtained diffraction image, the diffraction intensity was quantified using software mosflm (CCP4: Collaborative Computational Project No. 4) to determine the crystal structure factor.
- the final model contains two humanized Fab # 110 H 1 L 4 Fab ′ fragments and hCD 147 v 2 bound to each.
- the electron density corresponding to amino acid residue 23-203 was observed for one hCD147v2, while the electron density corresponding to domain 1 was not clear for the other, and the electron corresponding to amino acid residue 103-202. Only the density was observed.
- Example 18 Antitumor effect of humanized CD147 antibody in gastric cancer model With regard to human gastric cancer cell line KATO III cells (ATCC, Cat. HTB-103) of which CD147 positive was confirmed by a flow cytometer, anti-human CD147 human chimeric antibody and The antitumor effect of the humanized antibody was examined.
- human gastric cancer cell line KATO III cells ATCC, Cat. HTB-103 of which CD147 positive was confirmed by a flow cytometer, anti-human CD147 human chimeric antibody and The antitumor effect of the humanized antibody was examined.
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in FIG.
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- the effect was 31% in the LN22R8chIgG4p administration group and 54% in the # 110H1L4hIgG4P administration group.
- Example 19 Anti-Tumor Effect of Humanized CD147 Antibody in a Chronic Myeloid Leukemia Model The anti-tumor effect of human chronic myelogenous leukemia cell line KU812 cells (ATCC, Cat. CRL-2099) confirmed to be CD147 positive by flow cytometry The antitumor effect of human CD147 humanized antibody was examined.
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in FIG.
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- the effect was 48% in the imatinib administration group and 97% in the # 110H1L4hIgG4P administration group.
- Complete regression of the tumor was observed in 4 of 5 cases only in the # 110 H 1 L 4 h IgG 4 P administration group.
- a 5 ⁇ 10 6 human colon cancer cell line SW620 is suspended in 100% Matrigel (Corning Inc., Cat. 354234), and a 5-week example female NOD-scid mouse (NOD. CB17-Prkdc ⁇ scid> / J, CLEA Japan, Inc. (Sold separately) were implanted subcutaneously in the axillary region. Grouping was performed 3 days after transplantation based on the tumor volume, and 10 mg / kg of a humanized CD147 antibody (# 084 H 1 L 2 h Ig 4 P or # 110 H 1 L 4 h Ig 4 P) prepared by human chimeric CD 147 antibody (LN 22 R 8 ch IgG 4 P), Example 6-4-2.
- a humanized CD147 antibody # 084 H 1 L 2 h Ig 4 P or # 110 H 1 L 4 h Ig 4 P
- human chimeric CD 147 antibody LN 22 R 8 ch IgG 4 P
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in FIG.
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- Example 21 Antitumor Effect of Humanized CD147 Antibody in Renal Cancer 786-O Model The antitumor effect of the humanized antibody was examined for human renal cancer 786-O in which CD147 positive was confirmed by a flow cytometer. 5 ⁇ 10 6 cells of human kidney cancer 786-O are suspended with 50% Matrigel (Corning, Cat. 354234), and 5-week example female NOD-scid mice (NOD. CB17-Prkdc ⁇ scid> / J, Were implanted subcutaneously in the axilla region (purchased from CLEA Japan).
- Grouping was performed 3 days after transplantation based on the tumor volume, and 10 mg / kg of a humanized CD147 antibody (# 084 H 1 L 2 h Ig 4 P or # 110 H 1 L 4 h Ig 4 P) prepared by human chimeric CD 147 antibody (LN 22 R 8 ch IgG 4 P), Example 6-4-2.
- a humanized CD147 antibody # 084 H 1 L 2 h Ig 4 P or # 110 H 1 L 4 h Ig 4 P
- human chimeric CD 147 antibody LN 22 R 8 ch IgG 4 P
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in FIG.
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- Example 22 Antitumor Effect of Humanized CD147 Antibody in Acute Myeloid Leukemia (AML) Model Regarding human AML cell line OCI-AML3 cells (DSMZ, Cat. ACC 582) in which CD147 positive was confirmed by flow cytometer The antitumor effect of the humanized antibody was examined.
- AML Acute Myeloid Leukemia
- Tumor volume (mm 3 ) 1/2 ⁇ short diameter (mm) ⁇ short diameter (mm) ⁇ long diameter (mm)
- the results are shown in FIG.
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- Example 23 Activity Measurement of Humanized Anti-CD147 Antibody Having Different Binding Properties Suspension of 5 ⁇ 10 6 cells of human pancreatic strain MIA PaCa-2 in PBS containing 50% GFR-Matrigel (Corning Inc., Cat. 354230) 4 week-old female Nude mice (BALB / c Slc-nu / nu, purchased from Nippon SLC Co., Ltd.) were implanted subcutaneously in the axillary region.
- Three types of humanized CD147 antibodies (# 110H1L4hIgG4P, # 110H13L02hIgG4P and # 110H13L02hIgG4P with different binding to the CD147 protein prepared in Example 6) -4-2 were divided into groups based on tumor volume and 7 days after transplantation
- Gemcitabine purchased from Eli Lilly Japan
- the major axis and minor axis of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated by the following formula.
- the results are shown in FIG.
- the graph shows the mean value and the standard error together with the change of the tumor volume.
- the tumor growth inhibition rate of the control drug gemcitabine was 66%, whereas all humanized CD147 antibodies showed superior antitumor effect than gemcitabine in the 10 mg / kg administration group.
- HBS-EP + (manufactured by GE Healthcare Bioscience) was used as a running buffer, and CM5 (manufactured by GE Healthcare Bioscience) was used as a sensor chip.
- the diluted serial solution (0.5 to 8 ⁇ g / mL) of the antigen used in Example 2) -5 was flowed at 120 ⁇ L / min.
- the second addition was followed by monitoring of the dissociation phase for 300 seconds.
- 3 M magnesium chloride (manufactured by GE Healthcare Bioscience) was added for 30 seconds at a flow rate of 20 ⁇ L / min.
- the dissociation constant of the humanized CD147 antibody (# 084H1L2hIgG4P or # 110H1L4hIgG4P) was calculated by the method of Example 7) -1.
- Table 8 shows information on calculated dissociation constants, effector functions, and epitope regions.
- the # 84H1L2hIgG2 antibody was used for the # 084H1L2hIg4P antibody, and the # 110chIgG2 antibody was used for # 110H1L4hIg4P.
- Recombinant Human CD147 / Fc diluted with PBS was added to a 96-well plate (Thermo Scientific, Cat. 43454) at 2 ug / ml, 50 ul / well, and stored at 4 ° C. overnight. After removing the protein solution, 300 ul of PBS containing 1% BSA was added and warmed at room temperature for 1 hour. The solution was removed and 25 ul of existing antibody solution (0, 0.2, 2, 20 ug / ml) was added and warmed for 2 hours at room temperature.
- Binding of # 084 H 1 L 2 h Ig 4 P to Recombinant Human CD147 / Fc was inhibited in the presence of hIgG in the presence of 1 to 10 ug / ml of the existing CD147 antibody.
- the binding of # 110H1L4hIg4P to Recombinant Human CD147 / Fc was not inhibited in the presence of hIgG, 0.1-10 ug / ml of the existing CD147 antibody, but in the presence of # 110 chIgG2 antibody.
- Example 1-15 As a condition different from the method of -15, HepG2 cells (ATCC, Cat. HB-8065) are used as ADCC target cells, and # 110H1L4hIgG4P, # 084H1L2hIg4P, 4A5, 5F6, PPAT-082-03 as CD147 antibody. It was evaluated at a concentration of 1 ⁇ g / ml. The measurement was performed in triplicate, and the average value and the standard deviation were calculated. ADCC positive (+) when 5% or more of 51 Cr in cells was detected. If less than 5%, ADCC activity was negative (-). The results are shown in Table 8.
- # 110H1L4hIgG4P and # 084H1L2hIg4P were determined to be ADCC ( ⁇ ) without detection of ADCC activity. In 4A5, 5F6 and PPAT-082-03, 51 Cr of 5% or more of cells was detected, and it was regarded as ADCC positive (+).
- # 110H1L4hIgG4P, # 084H1L2hIg4P are ADCC negative and may induce cell death to normal cells such as blood cells expressing CD147 in human in vivo, but ADCC activity positive 4A5, 5F6, PPAT It is expected to be lower than -082-03.
- Example 1 As a condition different from the method of -16, human liver HepG2 cells (ATCC, Cat. HB-8065) are used as target cells, and # 110H1L4hIgG4P, # 084H1L2hIg4P, 4A5, 5F6 and PPAT as anti-human CD147 antibodies. Rabbit complement was added to a final concentration of 8% using -082-03 and measured. The measurement was performed in triplicate, and the mean value and the standard deviation were calculated.
- Example 1 As a condition different from the method of -17, human liver HepG2 cells (ATCC, Cat. HB-8065) are used as target cells, and # 110H1L4hIgG4P, # 084H1L2hIg4P, 4A5, 5F6, PPAT-082 as anti-CD147 antibody. -03 was added at a concentration of 1 ⁇ g / ml, and labeled RAW 264.7 cells were added in equal amounts of ADCP target cells, and ADCP activity was measured. The measurement was performed in triplicate, the mean value and the standard deviation were calculated, and the ADCP activity elevation was less than 10% weak positive ( ⁇ ) and 10% or more positive (+) positive than the human IgG treated group. It was shown to.
- the ADCP activity of # 110H1L4hIgG4P was less than 10% and was determined as ADCC activity ⁇ .
- the ADCP activity of the # 084 H 1 L 2 h IgG 4 P, 4 A 5, 5 F 6, and PPAT-08 2-03 antibodies was 10% or more, and was determined as ADCP activity +.
- # 110H1L4hIgG4P that recognizes CD147-D2 has lower ADCP activity than other CD147 antibodies that recognize CD147-D1.
- ADCP requires that the Fc portion of the antibody be recognized by the Fc ⁇ receptor expressed on macrophages and monocytes after CD147 antibody binds to CD147, but the epitope of # 110 antibody is on the cell surface
- the FC portion of the antibody bound to the antigen CD147 is less likely to be recognized by the Fc ⁇ receptor than other antibodies that recognize CD147-D1, and the ADCP activity may be lower.
- Possibility of inducing cell death to normal cells such as blood cells expressing CD147 in human beings with weak positive for # 110H1L4hIgG4P, ADCP activity, ADCP activity positive # 084H1L2hIg4P, 4A5, 5F6 , PPAT-082-03 is expected to be lower.
- HEL 92.1.7 cells purchased from ATCC, Cat. # TIB-180
- a 96-well U bottom plate (Sumitomo Bakelite, Cat. MS- 9096 U) at 1600 cells / 80 uL 10% FBS (high) clone Co., RPMI1640 medium (thermo Fisher Scientific containing Cat.SH30084.03), was added Cat.11875-093), 4 hours, 5% CO 2, 95% humidity, and cultured at 37 ° conditions .
- the CD147 antibody with cytocidal activity such as # 084 may cause toxicity such as thrombus via blood cell aggregation when administered to humans, so a dose of heparin or the like used for treatment of thrombus may be generated. It is desirable to avoid or reduce side effects by subcutaneous injection of low molecular weight heparin, or in combination with antiplatelet drugs.
- cytokine release syndrome With the administration of therapeutic antibodies, some antibodies such as OKT3 and TGN1412 activate immune cells to increase cytokines in the blood, resulting in severe cytokine release syndrome (Gaston, R., Kidney International, 1991, 141-148; Suntharalingam, G., et al., N. Engl. J. Med. 2006, 1018-1028). Some CD147 antibodies act on immune cells and have been reported to increase the production of interferon gamma and interleukin 4 (Hu, J., et al., J. Cell. Mol. Med., 2010, 2132-2143).
- the toxicity of antibody drug due to this cytokine release syndrome can be predicted by a cytokine release assay using peripheral blood (Vessillier, S. et al., J. Immunolol. Methods, 2015, 43-52).
- the risk of cytokine release syndrome was assessed by a similar human peripheral blood cytokine release assay.
- CD147 antibody # 110H1L4hIgG4P, # 110chIgG4ProFALA, # 084H1L2hIg4P, # 084H1L2hIg2 are used, and as comparative antibodies, bevacizumab (Genentech, Inc.), trattuzumab (Roche Pharma AG), alemtuzumab (Sanofi Co., Ltd.), anti-human CD3 antibody No. 317326) was used.
- # 110H1L4hIgG4P was administered to cynomolgus monkeys as an anti-CD147 antibody whose binding to human and monkey CD147 was confirmed by an experiment using a flow cytometer, and safety was evaluated.
- # 110H1L4hIgG4P As a result of single intravenous administration of # 110H1L4hIgG4P at 99.2 mg / kg, the maximum dose that can be administered to cynomolgus monkeys (1 male and 1 female each), the observation period up to 15 days after administration and histopathology at the end of the observation period No serious toxicities (changes in body weight and food consumption, histopathological changes) were observed on examination.
- # 110H1L4hIgG4P showed no toxicity to cynomolgus monkeys, indicating its potential to be used for human cancer treatment.
- Example 26 Effect of KLF5 on CD147 Antibody Susceptibility Lethal EMT signal is known as a signal of SMAD2 / SMAD3 / SMAD4 dependent cell death in cancer cells, and this Lethal EMT signal is an SMAD4 negative cancer cell. Reported that the expression of the transcription factor KLF5 protein, which is normally suppressed by the SMAD signal, increases and suppresses lethal signals (David, C. Cell, 2016, 1015-1030). The CD147 antibody of the present invention activated SMAD signal, and was considered to induce SMAD signal-dependent cell death because it exhibited an antitumor effect on SMAD4 positive cells. It was examined whether KLF5 is involved in the sensitivity of the CD147 antibody-dependent anti-tumor effect.
- KLF5 Expression Strain According to the method of Example 13, a KLF5 stable expression strain of MIA PaCa-2 cells was prepared. The amino acid and nucleotide sequences of human KLF5 are shown in SEQ ID NOs: 145 and 146, respectively.
- a retrovirus vector pQCXIP was prepared which incorporated the sequence (Refseq. ID: NM — 001730.4) contained in (Genscript, Cat. OH u 21 278 C) as the KLF 5 gene, and a retrovirus was used for production.
- a retrovirus was incorporated into the chromosome by viral infection, and puramycin resistant, KLF5 positive MIA PaCa-2 cells were selected and designated as KLF5 positive MIA PaCa-2 cells, MIA PaCa-2-KLF5.
- the retrovirus vector pQCXIP was similarly infected, and the puramycin resistant MIA PaCa-2 cells were designated as MIA PaCa-2-mock.
- CD147 antibody # 110 H1 L4 h IgG4 P The sensitivity to CD147 antibody # 110 H1 L4 h IgG4 P was compared.
- the humanized CD147 antibody (# 110H1L4hIg4P) prepared in Example 6) -4-2 was tail vein administered at 1 mg / kg to tumor-bearing mice (n 6). The antibody was similarly administered 7 days later.
- MIA PaCa-2-mock tumors had reduced the mean volume of the tumor to 9% of the control group, and were sensitive to the CD147 antibody.
- the average tumor volume of MIA PaCa-2-KLF5 was 80% of that of the control group, indicating low sensitivity to the CD147 antibody. It was found that the SMAD signal-dependent anti-tumor effect of the CD147 antibody was suppressed by the expression of KLF5.
- the present invention provides CD147-specific antibodies that activate CD147 and exhibit high anti-tumor effects.
- the present invention provides an antibody that exhibits high anti-tumor effects independently of effector function.
- the antibody of the present invention exhibits significantly higher efficacy in liver cancer cells than sorafenib, which is used as one of the standard therapeutic agents for liver cancer.
- the antibodies of the present invention exhibit significantly greater efficacy in pancreatic cancer cells than gemcitabine, which is used as one of the standard treatments for pancreatic cancer.
- the antibody of the present invention exhibits significantly higher efficacy in chronic myelogenous leukemia cells than imatinib, which is used as one of the standard therapeutic agents for chronic myelogenous leukemia.
- the anti-tumor effect of the antibody of the present invention does not depend on the effector function, and an anti-CD147 antibody excellent in safety with less concern in other safety endpoints is provided.
- CD147 is expressed not only on tumor cells but also on blood cells, the antibody of the present invention does not act on T cells and PBMCs and does not depend on effector function, so in development as an antitumor agent, It has the advantage of less safety concerns.
- the present invention provides a pharmaceutical composition comprising the above antibody, and a method of treating a tumor using the antibody and / or the pharmaceutical composition.
- SEQ ID NO: 1 Amino acid sequence of variant 1 of human CD 147
- SEQ ID NO: 2 nucleotide sequence of variant 1 of human CD 147
- SEQ ID NO: 3 amino acid sequence of variant 2 of human CD 147
- SEQ ID NO 4 nucleotide sequence of variant 2 of human CD 147
- 5 Amino acid sequence of human SMAD 4
- SEQ ID NO: 6 Nucleotide sequence of human SMAD 4
- SEQ ID NO 8 Amino acid sequence of light chain variable region of LN 22 R 8
- SEQ ID NO 9 LN 22 R 8
- SEQ ID NO: 141 amino acid sequence of # 131L2h SEQ ID NO: 142: nucleotide sequence of # 131 L2h SEQ ID NO: 143: mu3 region of human CD147 v 1 SEQ ID NO: 144: mu 3 region of cynomolgus monkey CD147 SEQ ID NO: 145: amino acid sequence of human KLF 5 SEQ ID NO: 146: human KLF 5 Nucleotide sequence SEQ ID NO: 147: # 110 H 13 h IgG 4 P amino acid sequence SEQ ID NO: 148: # 110 H 13 h IgG 4 P The nucleotide sequence SEQ ID NO 149: # 110L2h amino acid sequence SEQ ID NO 150: # 110L2h nucleotide sequence SEQ ID NO 151: # 110L12h amino acid sequence SEQ ID NO: 152: the # 110L12h nucleotide sequence
Abstract
Description
[1]
以下の(A)~(F)からなる群から選択される少なくともいずれか1つの抗体と、ヒトCD147への結合に対し競合し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(A)配列番号71に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号69に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(B)配列番号51に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号49に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(C)配列番号61に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号59に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(D)配列番号81に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号79に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(E)配列番号10に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号8に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、及び
(F)配列番号20に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号18に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体。
[2]
以下の(A)~(F)からなる群から選択される少なくともいずれか1つの抗体が結合するエピトープに結合し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(A)配列番号71に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号69に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(B)配列番号51に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号49に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(C)配列番号61に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号59に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(D)配列番号81に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号79に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(E)配列番号10に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号8に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、及び
(F)配列番号20に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号18に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体。
[3]
ADCC活性が低下又は欠失した、[1]又は[2]に記載のヒトCD147抗体又は該抗体の抗原結合断片。
[4]
CDC活性が低下又は欠失した、[1]~[3]のいずれか1項に記載のヒトCD147抗体又は該抗体の抗原結合断片。
[5]
ADCP活性が低下又は欠失した、[1]~[4]のいずれか1項に記載のヒトCD147抗体又は該抗体の抗原結合断片。
配列番号3の106番目のアルギニン(Arg)から165番目のグリシン(Gly)の残基を含むエピトープに結合する、[1]~[5]のいずれか1項に記載の抗体又は該抗体の抗原結合断片。
[7]
配列番号3に記載のアミノ酸配列中の、106番目のアルギニン(Arg)、108番目のリシン(Lys)、109番目のアラニン(Ala)、110番目のバリン(Val)、127番目のリシン(Lys)、128番目のセリン(Ser)、129番目のグルタミン酸(Glu)、130番目のセリン(Ser)、131番目のバリン(Val)、132番目のプロリン(Pro)、133番目のプロリン(Pro)、134番目のバリン(Val)、164番目のグルタミン(Gln)及び165番目のグリシン(Gly)の各残基を含むエピトープに結合する、[1]~[6]のいずれか1項に記載の抗体又は該抗体の抗原結合断片。
[8]
重鎖配列が、CDRH1、CDRH2及びCDRH3を有する可変領域を含み、前記CDRH1は配列番号75に示されるアミノ酸配列からなり、前記CDRH2は配列番号76に示されるアミノ酸配列からなり、前記CDRH3は、配列番号77に示されるアミノ酸配列からなること;及び
軽鎖配列がCDRL1、CDRL2及びCDRL3を有する可変領域を含み、前記CDRL1は配列番号72に示されるアミノ酸配列からなり、前記CDRL2は配列番号73に示されるアミノ酸配列からなり、前記CDRL3は、配列番号74に示されるアミノ酸配列からなること;
を特徴とする、[1]~[7]のいずれか1項に記載の抗体又は該抗体の抗原結合断片。
[9]
配列番号143に記載のアミノ酸配列、又は、配列番号143の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列を含むエピトープに結合する、[1]~[5]のいずれか1項に記載の抗体又は該抗体の抗原結合断片。
[10]
重鎖配列が、CDRH1、CDRH2及びCDRH3を有する可変領域を含み、前記CDRH1は配列番号55に示されるアミノ酸配列からなり、前記CDRH2は配列番号56に示されるアミノ酸配列からなり、前記CDRH3は、配列番号57に示されるアミノ酸配列からなること;及び
軽鎖配列がCDRL1、CDRL2及びCDRL3を有する可変領域を含み、前記CDRL1は配列番号52に示されるアミノ酸配列からなり、前記CDRL2は配列番号53に示されるアミノ酸配列からなり、前記CDRL3は、配列番号54に示されるアミノ酸配列からなること;
を特徴とする、[1]~[5]又は9のいずれか1項に記載の抗体又は該抗体の抗原結合性断片。
重鎖配列が、CDRH1、CDRH2及びCDRH3を有する可変領域を含み、前記CDRH1は配列番号65に示されるアミノ酸配列からなり、前記CDRH2は配列番号66に示されるアミノ酸配列からなり、前記CDRH3は、配列番号67に示されるアミノ酸配列からなること;及び
軽鎖配列がCDRL1、CDRL2及びCDRL3を有する可変領域を含み、前記CDRL1は配列番号62に示されるアミノ酸配列からなり、前記CDRL2は配列番号63に示されるアミノ酸配列からなり、前記CDRL3は、配列番号64に示されるアミノ酸配列からなること;
を特徴とする、[1]~[5]又は9のいずれか1項に記載の抗体又は該抗体の抗原結合性断片。
[12]
重鎖配列が、CDRH1、CDRH2及びCDRH3を有する可変領域を含み、前記CDRH1は配列番号85に示されるアミノ酸配列からなり、前記CDRH2は配列番号86に示されるアミノ酸配列からなり、前記CDRH3は、配列番号87に示されるアミノ酸配列からなること;及び
軽鎖配列がCDRL1、CDRL2及びCDRL3を有する可変領域を含み、前記CDRL1は配列番号82に示されるアミノ酸配列からなり、前記CDRL2は配列番号83に示されるアミノ酸配列からなり、前記CDRL3は、配列番号84に示されるアミノ酸配列からなること;
を特徴とする、[1]~[5]又は9のいずれか1項に記載の抗体又は該抗体の抗原結合性断片。
[13]
Fab、F(ab’)2、Fab’及びFvからなる群から選択される、[1]~[12]のいずれか1項に記載の抗体の抗原結合断片。
[14]
scFvであることを特徴とする、[1]~[12]のいずれか1項に記載の抗体。
[15]
キメラ抗体であることを特徴とする、[1]~[12]のいずれか1項に記載の抗体文は該抗体の抗原結合断片。
ヒ卜化されていることを特徴とする、[1]~[12]のいずれか1項に記載の抗体文は該抗体の抗原結合断片。
[17]
重鎖がヒ卜免疫グ口ブリンG1重鎖、ヒ卜免疫グ口ブリンG2重鎖又はヒ卜免疫グ口ブリンG4重鎖の定常領域を含み、軽鎖がヒ卜免疫グ口プリンκ軽鎖の定常領域を含む、[1]~[16]のいずれか1項に記載の抗体。
[18]
重鎖がヒ卜免疫グ口ブリンG4重鎖の定常領域を含む、[17]に記載の抗体。
[19]
ヒ卜免疫グ口ブリンG4重鎖の定常領域において、EUインデックスにより示される228番目のセリン(Ser)がプ口リン(Pro)により置換された、[18]に記載の抗体。
[20]
ヒ卜免疫グ口ブリンG4重鎖の定常領域において、EUインデックスにより示される234番目のフェニルアラニン(Phe)がアラニン(Ala)へ置換され、235番目のロイシン(Leu)がアラニン(Ala)に置換されている、[18]の抗体。
ヒ卜免疫グ口ブリンG4重鎖の定常領域において、EUインデックスにより示される228番目のセリン(Ser)がプ口リン(Pro)により置換され、234番目のフェニルアラニン(Phe)がアラニン(Ala)へ置換され、235番目のロイシン(Leu)がアラニン(Ala)に置換されている、[18]の抗体。
[22]
重鎖がヒ卜免疫グ口プリンG2重鎖の定常領域を含む、[17]に記載の抗体。
[23]
以下の(c)及び(d)を有し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(c)以下の(c1)~(c4)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(c1)配列番号135に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域;
(c2)配列番号147に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域;
(c3)(c1)又は(c2)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(c4)(c1)~(c3)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(d)以下の(d1)~(d5)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(d1)配列番号137に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(d2)配列番号149に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(d3)配列番号151に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(d4)(d1)~(d3)のいずれか1に記載の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(d5)(d1)~(d4)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。
[24]
配列番号135に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号137に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、[23]に記載の抗体又は該抗体の抗原結合性断片。
配列番号135に示されるアミノ酸配列の20~463番目のアミノ酸残基からなる重鎖、及び、配列番号137に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、[23]に記載の抗体又は該抗体の抗原結合性断片。
[26]
配列番号147に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号149に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、[23]に記載の抗体又は該抗体の抗原結合性断片。
[27]
配列番号147に示されるアミノ酸配列の20~463番目のアミノ酸残基からなる重鎖、及び、配列番号149に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、[23]に記載の抗体又は該抗体の抗原結合性断片。
[28]
配列番号147に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号151に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、[23]に記載の抗体又は該抗体の抗原結合性断片。
[29]
配列番号147に示されるアミノ酸配列の20~463番目のアミノ酸残基からなる重鎖、及び、配列番号151に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、[23]に記載の抗体又は該抗体の抗原結合性断片。
[30]
以下の(a)及び(b)を有し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(a)以下の(a1)~(a4)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(a1)配列番号123に示されるアミノ酸配列の20~140番目のアミノ酸残基からなる重鎖可変領域;
(a2)配列番号125に示されるアミノ酸配列の20~140番目のアミノ酸残基からなる重鎖可変領域;
(a3)(a1)又は(a2)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(a4)(a1)~(a3)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(b)以下の(b1)~(b3)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(b1)配列番号127に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(b2)(b1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(b3)(b1)又は(b2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。
配列番号123に示されるアミノ酸配列の20~140番目のアミノ酸残基からなる重鎖可変領域又は配列番号125に示されるアミノ酸配列の20~140番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号127に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、[30]に記載の抗体又は該抗体の抗原結合性断片。
[32]
配列番号123に示されるアミノ酸配列の20~466番目のアミノ酸残基からなる重鎖又は配列番号125に示されるアミノ酸配列の20~467番目のアミノ酸残基からなる重鎖、及び、配列番号127に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、[30]に記載の抗体又は該抗体の抗原結合性断片。
[33]
以下の(e)及び(f)を有し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(e)以下の(e1)~(e4)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(e1)配列番号129に示されるアミノ酸配列の20~137番目のアミノ酸残基からなる重鎖可変領域;
(e2)配列番号131に示されるアミノ酸配列の20~137番目のアミノ酸残基からなる重鎖可変領域;
(e3)(e1)又は(e2)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(e4)(e1)~(e3)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(f)以下の(f1)~(f3)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(f1)配列番号133に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(f2)(f1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(f3)(f1)又は(f2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。
[34]
配列番号129に示されるアミノ酸配列の20~137番目のアミノ酸残基からなる重鎖可変領域又は配列番号131に示されるアミノ酸配列の20~137番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号133に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、[33]に記載の抗体又は該抗体の抗原結合性断片。
[35]
配列番号129に示されるアミノ酸配列の20~463番目のアミノ酸残基からなる重鎖又は配列番号131に示されるアミノ酸配列の20~464番目のアミノ酸残基からなる重鎖、及び、配列番号133に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、[33]に記載の抗体又は該抗体の抗原結合性断片。
以下の(g)及び(h)を有し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(g)以下の(g1)~(g3)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(g1)配列番号139に示されるアミノ酸配列の20~138番目のアミノ酸残基からなる重鎖可変領域;
(g2)(g1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(g3)(g1)又は(g2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(h)以下の(h1)~(h3)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(h1)配列番号141に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(h2)(h1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(h3)(h1)又は(h2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。
[37]
配列番号139に示されるアミノ酸配列の20~138番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号141に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、[36]に記載の抗体又は該抗体の抗原結合性断片。
[38]
配列番号139に示されるアミノ酸配列の20~464番目のアミノ酸残基からなる重鎖、及び、配列番号141に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、[36]に記載の抗体又は該抗体の抗原結合性断片。
[39]
ADCC活性が低下又は欠失した、[23]~[38]のいずれか1項に記載のヒトCD147抗体又は該抗体の抗原結合断片。
[40]
CDC活性が低下又は欠失した、[23]~[39]のいずれか1項に記載のヒトCD147抗体又は該抗体の抗原結合断片。
ADCP活性が低下又は欠失した、[23]~[40]のいずれか1項に記載のヒトCD147抗体又は該抗体の抗原結合断片。
[42]
[1]~[41]のいずれか1項に記載の抗体又は該抗体の抗原結合断片の少なくともいずれか一つを含有することを特徴とする、医薬組成物。
[43]
抗腫瘍剤である、[42]に記載の医薬組成物。
[44]
腫瘍が、CD147を発現する腫瘍である、[43]に記載の医薬組成物。
[45]
腫瘍が、膵臓癌、肝癌、胃癌、大腸癌、腎癌、乳癌、子宮癌、卵巣癌、肺癌、リンパ腫、甲状腺癌、皮膚癌、頭頸部癌、肉腫、前立腺癌、膀胱癌、脳腫瘍、消化管間質腫瘍(GIST)、白血病、急性骨髄性白血病(AML)、慢性骨髄性白血病(CML)、慢性リンパ性白血病(CLL)、急性リンパ性白血病(ALL)、悪性リンパ腫、B細胞リンパ腫、非ホジキンリンパ腫又はびまん性大細胞型B細胞リンパ腫(DLBCL)である、[43]又は[44]に記載の医薬組成物。
腫瘍が、膵臓癌、肝癌、胃癌、大腸癌、腎癌、白血病、急性骨髄性白血病(AML)、慢性骨髄性白血病(CML)、慢性リンパ性白血病(CLL)、急性リンパ性白血病(ALL)、悪性リンパ腫、B細胞リンパ腫、非ホジキンリンパ腫又はびまん性大細胞型B細胞リンパ腫(DLBCL)である、[43]~[45]のいずれか1項に記載の医薬組成物。
[47]
腫瘍がSMAD4陽性の腫瘍又はKLF5の発現が低下又は欠失した腫瘍である、[43]~[46]のいずれか1項に記載の医薬組成物。
[48]
さらに別の抗腫瘍剤を含有する、[42]~[47]のいずれか1項に記載の医薬組成物。
[49]
[1]~[41]のいずれか1項に記載の抗体若しくは該抗体の抗原結合性断片又は[42]~[48]のいずれか1項に記載の医薬組成物を患者に投与することを特徴とする腫瘍の治療方法。
[50]
腫瘍が、CD147を発現する腫瘍である、[49]に記載の治療方法。
腫瘍が、膵臓癌、肝癌、胃癌、大腸癌、腎癌、乳癌、子宮癌、卵巣癌、肺癌、リンパ腫、甲状腺癌、皮膚癌、頭頸部癌、肉腫、前立腺癌、膀胱癌、脳腫瘍、消化管間質腫瘍(GIST)、白血病、急性骨髄性白血病(AML)、慢性骨髄性白血病(CML)、慢性リンパ性白血病(CLL)、急性リンパ性白血病(ALL)、悪性リンパ腫、B細胞リンパ腫、非ホジキンリンパ腫又はびまん性大細胞型B細胞リンパ腫(DLBCL)である、[49]又は[50]のいずれか1項に記載の治療方法。
[52]
腫瘍が、膵臓癌、肝癌、胃癌、大腸癌、腎癌、白血病、急性骨髄性白血病(AML)、慢性骨髄性白血病(CML)、慢性リンパ性白血病(CLL)、急性リンパ性白血病(ALL)、悪性リンパ腫、B細胞リンパ腫、非ホジキンリンパ腫又はびまん性大細胞型B細胞リンパ腫(DLBCL)である、[49]~[51]に記載の治療方法。
[53]
腫瘍がSMAD4陽性の腫瘍又はKLF5の発現が低下又は欠失した腫瘍である、[49]~[52]のいずれか1項に記載の治療方法。
[54]
別の抗腫瘍剤と組み合わせて投与される、[49]~[53]のいずれか1項に記載の治療方法。
[55]
[1]~[41]のいずれか1項に記載の抗体又は当該抗体の機能性断片をコードするポリヌクレオチド。
以下の(j1)~(j3)からなる群から選択されるいずれか1に記載のポリヌクレオチドを含む[55]に記載のポリヌクレオチド:
(j1)配列番号75に記載のアミノ酸配列からなるCDRH1、配列番号76に記載のアミノ酸配列からなるCDRH2及び配列番号77に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに、配列番号72に記載のアミノ酸配列からなるCDRL1、配列番号73に記載のアミノ酸配列からなるCDRL2及び配列番号74に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、
(j2)(j1)に記載のヌクレオチド配列と少なくとも95%の配列同一性を有するポリヌクレオチド;及び
(j3)(j1)又は(j2)に記載のポリヌクレオチドにおいて1~数個のヌクレオチドが置換、欠失又は付加されたポリヌクレオチド。
[57]
以下の(i1)~(i3)からなる群から選択されるいずれか1に記載のポリヌクレオチドを含む[55]に記載のポリヌクレオチド:
(i1)配列番号55に記載のアミノ酸配列からなるCDRH1、配列番号56に記載のアミノ酸配列からなるCDRH2及び配列番号57に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに、配列番号52に記載のアミノ酸配列からなるCDRL1、配列番号53に記載のアミノ酸配列からなるCDRL2及び配列番号54に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド;
(i2)(i1)に記載のヌクレオチド配列と少なくとも95%の配列同一性を有するポリヌクレオチド;及び
(i3)(i1)又は(i2)に記載のポリヌクレオチドにおいて1~数個のヌクレオチドが置換、欠失又は付加されたポリヌクレオチド。
[58]
以下の(k1)~(k3)からなる群から選択されるいずれか1に記載のポリヌクレオチドを含む[55]に記載のポリヌクレオチド:
(k1)配列番号65に記載のアミノ酸配列からなるCDRH1、配列番号66に記載のアミノ酸配列からなるCDRH2及び配列番号67に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに、配列番号62に記載のアミノ酸配列からなるCDRL1、配列番号63に記載のアミノ酸配列からなるCDRL2及び配列番号64に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、
(k2)(k1)に記載のヌクレオチド配列と少なくとも95%の配列同一性を有するポリヌクレオチド;及び
(k3)(k1)又は(k2)に記載のポリヌクレオチドにおいて1~数個のヌクレオチドが置換、欠失又は付加されたポリヌクレオチド。
[59]
以下の(m1)~(m3)からなる群から選択されるいずれか1に記載のポリヌクレオチドを含む[55]に記載のポリヌクレオチド:
(m1)配列番号85に記載のアミノ酸配列からなるCDRH1、配列番号86に記載のアミノ酸配列からなるCDRH2及び配列番号87に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに、配列番号82に記載のアミノ酸配列からなるCDRL1、配列番号83に記載のアミノ酸配列からなるCDRL2及び配列番号84に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、
(m2)(m1)に記載のヌクレオチド配列と少なくとも95%の配列同一性を有するポリヌクレオチド;及び
(m3)(m1)又は(m2)に記載のポリヌクレオチドにおいて1~数個のヌクレオチドが置換、欠失又は付加されたポリヌクレオチド。
[60]
[55]~[59]のいずれか1項に記載のポリヌクレオチドを含有する発現ベクター。
[60]に記載の発現ベクターにより形質転換された宿主細胞。
[62]
[61]に記載の宿主細胞を培養し、培養産物から目的の抗体又は当該抗体の機能性断片を採取する工程を含む、[1]~[41]のいずれか1項に記載の抗体又は当該抗体の機能性断片の製造方法。
[63]
CD147を介したシグナル伝達の活性化が、p38の活性化及び/又はSMAD4の活性化である、[1]~[41]のいずれか1項に記載の抗体又は当該抗体の機能性断片。
[64]
p38MAPKの活性化及び/又はSMAD4の活性化が、p38MAPKの発現量の増加、p38MAPKのリン酸化、HSP27のリン酸化、CXCL8発現量の増加、rhoB発現量の増加、KLF5 mRNAの低下又はKLF5蛋白質発現量の低下である、[63]に記載の抗体又は当該抗体の機能性断片。
[65]
[63]又は[64]に記載の抗体又は該抗体の抗原結合性断片を投与することを特徴とする腫瘍の治療方法。
癌患者由来の生物学的試料を用い、該生物学的試料中に含まれるSMAD4の発現又はKLF5の発現を検出し、SMAD4が検出された患者又はKLF5の発現低下若しくは欠失が検出された患者を、[1]~[41]のいずれか1項に記載の抗体若しくは当該抗体の機能性断片又は[42]~[48]のいずれか1項に記載の医薬組成物による癌の治療への応答性があると判定することを含む、癌の治療への応答性を予測する方法。
[67]
癌患者由来の生物学的試料を用い、該生物学的試料中におけるSMAD4の発現の有無又はKLF5の発現を検出し、SMAD4が検出された患者又はKLF5の発現低下若しくは欠失が検出された患者を、[1]~[41]のいずれか1項に記載の抗体若しくは当該抗体の機能性断片又は[42]~[48]のいずれか1項に記載の医薬組成物による癌の治療の対象者として選別することを含む、癌の治療の対象を選別する方法。
[68]
癌患者由来の生物学的試料を用い、該生物学的試料中に含まれるSMAD4の発現の有無又はKLF5の発現を検出し、SMAD4が検出された患者又はKLF5の発現低下若しくは欠失が検出された患者に対し、[1]~[41]のいずれか1項に記載の抗体若しくは当該抗体の機能性断片又は[42]~[48]のいずれか1項に記載の医薬組成物を投与することを含む、癌の治療方法。
[69]
[1]~[41]のいずれか1項に記載の抗体若しくは当該抗体の機能性断片又は[42]~[48]のいずれか1項に記載の医薬組成物による癌の治療への応答性を判定するためのキットであって、癌患者由来の生物学的試料中のSMAD4の発現又はKLF5の発現を検出する手段を少なくとも含む、キット。
[70]
[1]~[41]のいずれか1項に記載の抗体又は該抗体の抗原結合断片と他の薬物がコンジュゲートした、抗体薬物複合体。
[1]~[41]のいずれか1項に記載の抗体の抗原結合断片と、CD147以外の抗原に結合する抗原結合断片を含む、バイスペシフィック抗体。
補体依存性細胞傷害(CDC)活性は、血液に含まれる補体、抗体および標的細胞を接触させた場合に生じる細胞死を測定することで評価ができる。本発明のヒトCD147抗体のCDC活性は、次のように測定される。評価するヒトCD147抗体による補体依存的な殺細胞活性(CDC活性)を標的細胞としてヒト膵臓株MIA PaCa-2を用いて評価する。補体として市販のウサギ補体(Low Tox-M Rabbit Complement、CEDARLANE LABORATORIES LIMITED、Cat.CL3051)を用いる。CDC活性陰性の対照抗体としてヒトIgG(hIgG,ChromPure Human IgG,Jackson ImmunoResearch Laboratories社,Cat.009-000-003)を用いる。評価する抗体及び陰性の対象抗体を、それぞれ、0、0.1、1又は、10μg/mlの濃度で1時間、4℃で処理した後、ウサギ補体を終濃度7.5%となるように添加し、37℃、5%CO2存在下で、3時間加温後、生細胞に含まれる細胞内ATPをCellTiter-Glo Lumimescent Cell Viability Assay(Promega社,Cat.G7572)を用いて測定した。CellTiter-Glo Lumimescent Cell Viability Assay を用いて得られる発光シグナルについて、EnVision 2104 Multilabel Reader (Perkin Elmer社)を用いて定量する。測定は3重に実施し、平均値と標準偏差を算出する。無処理の細胞から得られた発光シグナルを100%として、抗体と補体依存的に減少した発光シグナルをCDC活性とする。
CD147は、イムノグロブリン様ドメインを2~3有する1回膜貫通蛋白質であり、CD147同士の相互作用、CD44、Integrinファミリー分子、CD98、VEGFR、CypA/B、MCT1/3/4といった増殖、浸潤、炎症に関与する細胞外や細胞膜表面の分子と相互作用することで、下流シグナル関連分子、FAK、MEK、Erk、JAK/STAT、AKT、MAPKファミリー分子を活性化させ、MMPをはじめとするプロテアーゼ産生、癌の増殖、転移、浸潤を促すことが知られている。
本発明のCD147に対する抗体は、非ヒト動物を目的抗原で免疫し、免疫成立後の動物からリンパ液、リンパ組織、血球試料又は骨髄由来の細胞を採取し、公知の方法(例えば、Kohler and Milstein,Nature(1975)256,p.495-497、Kennet,R.ed.,Monoclonal Antibodies,p.365-367,Plenum Press,N.Y.(1980))に従って、CD147に対する抗体を産生する抗体産生細胞とミエローマ細胞とを融合させることによりハイブリドーマを樹立し、モノクローナル抗体を得ることができる。このような方法の具体的な例は、WO2009/48072(2009年4月16日公開)及びWO2010/117011(2010年10月14日公開)に記載されている。このようにして得られたモノクローナル抗体の例としては、例えば、LN22R8、2P10F2、rat_CD147_#84、rat_CD147_#101、rat_CD147_#110又はrat_CD147_#131を挙げることができる。しかし、モノクローナル抗体を取得する方法は、既に確立された分野に該当し、前記の具体例に限定されるものではない。
(a)以下の(a1)乃至(a4)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(a1)配列番号123に示されるアミノ酸配列の20乃至140番目のアミノ酸残基からなる重鎖可変領域;
(a2)配列番号125に示されるアミノ酸配列の20乃至140番目のアミノ酸残基からなる重鎖可変領域;
(a3)(a1)又は(a2)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(a4)(a1)乃至(a3)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(b)以下の(b1)乃至(b3)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(b1)配列番号127に示されるアミノ酸配列の21乃至128番目のアミノ酸残基からなる軽鎖可変領域;
(b2)(b1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(b3)(b1)又は(b2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。
(e)以下の(e1)乃至(e4)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(e1)配列番号129に示されるアミノ酸配列の20乃至137番目のアミノ酸残基からなる重鎖可変領域;
(e2)配列番号131に示されるアミノ酸配列の20乃至137番目のアミノ酸残基からなる重鎖可変領域;
(e3)(e1)又は(e2)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(e4)(e1)乃至(e3)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(f)以下の(f1)乃至(f3)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(f1)配列番号133に示されるアミノ酸配列の21乃至128番目のアミノ酸残基からなる軽鎖可変領域;
(f2)(f1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(f3)(f1)又は(f2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。
(c)以下の(c1)~(c4)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(c1)配列番号135に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域;
(c2)配列番号147に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域;
(c3)(c1)又は(c2)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(c4)(c1)~(c3)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(d)以下の(d1)~(d5)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(d1)配列番号137に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(d2)配列番号149に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(d3)配列番号151に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(d4)(d1)~(d3)のいずれか1に記載の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(d5)(d1)~(d4)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。
(g)以下の(g1)~(g3)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(g1)配列番号139に示されるアミノ酸配列の20~138番目のアミノ酸残基からなる重鎖可変領域;
(g2)(g1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(g3)(g1)又は(g2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(h)以下の(h1)~(h3)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(h1)配列番号141に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(h2)(h1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(h3)(h1)又は(h2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。
上述の「抗CD147抗体の製造」の項に記載された方法で得られる抗CD147抗体の中から、本発明の抗CD147抗体を得ることができる。このようにして得られた抗体は、腫瘍及び/又は癌の治療及び/又は予防剤として用いることができる。 本発明の抗CD147抗体は、優れた抗腫瘍活性を有し、腫瘍又は癌の治療薬として有用である。本発明の抗CD147抗体は、ゲムシタビン耐性癌細胞やソラフェニブ低感受性癌細胞に対しても優れた抗腫瘍効果を示した。本発明の抗CD147抗体は、慢性骨髄性白血病細胞において、イマチニブよりも顕著に強い薬効を示した。
1)-1 CD147発現ベクターの作製
市販のヒトCD147遺伝子(BSG variant2/CD147v2)のクローンIOH3378(インビトロジェン社)と哺乳類細胞用発現ベクターpcDNA-DEST40(インビトロジェン社)をGateway LR クロナーゼを用いて反応させ、ヒトCD147v2発現ベクター(pcDNA-DEST40-CD147v2)を作製した。
4~6週齢のBALB/cAnNCrlCrljマウス(日本チャールス・リバー社)を使用した。0日目、7日目、15日目及び24日目にベルセン(Thermo Fisher Scientific社)で剥がした5×106個のLNCaP細胞(ATCC,CRL-1740)をPBSに懸濁して背部皮下に投与した。31日目に同じ細胞を5×106個静脈投与し、同日に脾臓を採取しハイブリドーマ作製に用いた。脾臓細胞とマウスミエローマP3X63Ag8U.1細胞(ATCC,CRL-1597)とをPEG4000(IBL社)を用いて細胞融合しハイブリドーマを作製した。ハイブリドーマの単離、培養には、ClonaCell-HY MediumD(STEMCELL TECHNOLOGIES社)、ClonaCell-HY MediumE(STEMCELL TECHNOLOGIES社)を用いた。
7週齢のWKY/Izm(日本エスエルシー株式会社)を使用した。ヒト膵臓癌細胞株PANC-1を1×107個、臀部に免疫し13日後に腸骨リンパ節細胞を採取しハイブリドーマ作製に用いた。ラット脾臓細胞とマウスミエローマSP2/0-Ag14細胞(ATCC, CRL-1581)をLF301細胞融合装置(株式会社ベックス)を用いて細胞融合しハイブリドーマを作製した。ハイブリドーマの単離、培養には、ClonaCell-HY MediumD(STEMCELL TECHNOLOGIES社)、ClonaCell-HY MediumE(STEMCELL TECHNOLOGIES社)を用いた。
ヒトCD147Fc融合蛋白質(Sino Biological社、Cat.10186-H02H)とマウスCD147Fc融合蛋白質(Sino Biological社、Cat.50332-M03H)を使用した。ヒトCD147Fc融合蛋白質とマウスCD147Fc融合蛋白質は、PBS緩衝液を添加し、氷上で溶解し、1μg/mlに調製した。同蛋白質溶解液を96ウェルプレート(NUNC社、Cat.442404)に100μl加え、4℃一晩保存し、ウェルをCD147Fc融合蛋白質でコートした。蛋白質溶解液を除き、1%BSA(Research Organics社、Cat.1334A)を含むPBS緩衝液でウェルを4℃、2時間ブロッキングした。0.05%Tween20(ATTO社、Cat.WSE-7235)を含むPBS緩衝液でウェルを3回洗浄したのち、実施例1)-2、1)-3で調製したハイブリドーマ培養上清をPBS緩衝液で20倍に希釈し各ウェルに加え室温で1時間加温した。0.05%Tween20(ATTO社、Cat.WSE-7235)を含むPBS緩衝液でウェルを3回洗浄した後、1%BSAを含むPBS緩衝液で50000倍に希釈したanti-rat-Fab2-igG-HRP(Jackson ImmunoResearch社、Cat.112-036-072)を100μl加え30分間、室温で振盪した。0.05%Tween20(ATTO社、Cat.WSE-7235)を含むPBS緩衝液でウェルを5回洗浄した後、100μlのHRP酵素発色試薬(eBioscience社、Super AquaBlue ELISA substrate,Cat.00-4203)を加え、10~20分室温で加温し、プレートリーダー(Envision、パーキンエルマー社)で405nmの吸光度を測定した。2~3ウェルの吸光度の測定値について平均値を算出し、1次抗体なしの対照ウェルでの測定値の2倍以上の吸光度が観察された抗体について結合性あり(+)、2倍に満たないものを結合性なし(-)と判定し表1にまとめた。LN22R8、2P1A6、2P3A9、2P3G8、2P8C12、2P10F2、2P2D7、2P2D10、2P1B7の培養上清について、ヒトCD147Fc融合蛋白質コートウェル特異的な発色を確認した。LN24R7、2P5F5、2P6A2、2P3G8については、ヒト及びマウスCD147Fc融合蛋白質コートウェル特異的な発色を確認した。
実施例1)-4で抗ヒトCD147抗体産生が確認されたハイブリドーマで、安定的に培養可能なものについて、市販のIsotypingキットを用いて、培養上清に含まれる抗体のアイソタイプを決定し表2に示した。CL-1000フラスコ(日本ベクトン・ディッキンソン株式会社)を用いて、これらのハイブリドーマを培養し、モノクローナル抗体を含むハイブリドーマ培養上清を調製した。
実施例1)-5で作製した培養上清から抗体を精製した。抗ヒトCD147マウスモノクローナル抗体については、rProtein Aアフィニティークロマトグラフィー(4~6℃下)1段階工程で精製した。rProtein Aアフィニティークロマトグラフィー精製後のバッファー置換工程は4~6℃下で実施した。最初に培養上清をPBSで平衡化したMabSelectSuRe(GE Healthcare Bioscience社製)が充填されたカラムにアプライした。培養液がカラムに全て入ったのち、カラム容量2倍以上のPBSでカラムを洗浄した。次に2M アルギニン塩酸塩溶液(pH4.0)で溶出し、抗体の含まれる画分を集めた。その画分を透析(Thermo Scientific社、Slide-A-Lyzer Dialysis Cassette)によりHBSor(25mM Histidine/5% Sorbitol/pH6.0)への液置換を行った。Centrifugal UF Filter Device VIVASPIN20(分画分子量UF10K,Sartorius社,4℃下)にて濃縮し、IgG濃度を4.9mg/mlに調製した。最後にMinisart-Plus filter(Sartorius社)でろ過し、精製サンプルとした。
1×107個のヒト膵臓株PANC-1をPBSで懸濁し、NOD-scidマウス(日本チャールス・リバー社、NOD.CB17-Prkdc<scid>/J)の腋窩部皮下に移植した。腫瘍体積をもとに群分けをし、移植の27、34、41日後にマウス抗CD147抗体(LN22R8)、ラット抗CD147抗体(2P1A6、2P1B7、2P3G8、2P2D10、2P8C12、2P10F2)を10mg/kgで担癌マウスの腹腔内に投与した(n=6)。移植の27、34日後にラット抗CD147抗体(2P2D6)を10mg/kgで担癌マウスの腹腔内に投与した(n=6)。移植腫瘍の長径及び短径を週2回、電子デジタルノギス(株式会社ミツトヨ製)を用いて測定し、以下に示す計算式により腫瘍体積を算出した。
結果を図1(a)~(c)に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。2P2D6抗体、2P3G8抗体及び2P2D10抗体の結果を図1(a)に示した。最終測定日である移植48日後における腫瘍増殖抑制率は、10mg/kg投与群で、それぞれ、10%、45%及び40%であった。LN22R8抗体、2P1A6抗体及び2P1B7抗体の結果を図1(b)に示した。最終測定日である移植48日後における腫瘍増殖抑制率は、10mg/kg投与群で、それぞれ、50%、26%及び24%であった。2P8C12抗体及び2P10F2抗体の結果を図1(c)に示した。最終測定日である移植48日後における腫瘍増殖抑制率は、10mg/kg投与群で、それぞれ、-2%及び62%であった。
CHO-K1細胞(ATCC、CCL-61)にLipofectamine 2000(Thermofishers scientific社、Cat.11668-019)を用いて、実施例1)-1で作製したpcDNA-DEST40-CD147v2、又はpCMV3-cynoBSGを導入し、1日後にマウス抗ヒトCD147抗体(LN22R8)、又はラット抗ヒトCD147抗体(2P1A6、2P1B7、2P3G8、2P2D10、2P8C12、2P10F2、2P2D6)、10μg/mlで処理し、抗マウスIgG-FITC(MP Biomedical社、Cat.554936)又は、抗ラットIgG-PE(BD Biosciences社、Cat.550767)を用いて各抗体のCD147発現CHO-K1細胞への結合を蛍光検出可能にした。CHO-K1細胞のヒトとカニクイザルCD147発現は、市販の抗CD147抗体(MEM-M6/1、Ab serotec社、 Cat.MCA28822)の結合により蛍光検出可能にした。上記の細胞について、フローサイトメーター(CantoII、BD Bioscience社)の測定を実施し、図2-1~3に結果をまとめた。図の縦軸は細胞数を示し、横軸は蛍光シグナルの強度を示す。
エピトープ解析用のヒトCD147異体発現ベクターの作製
カニクイザルとヒトのCD147のアミノ酸配列は、BLAST検索(https://blast.ncbi.nlm.nih.gov/Blast.cgi)の結果、81%が同一であり、限られたアミノ酸の違いが、CD147抗体結合性のエピトープ認識に直接影響していると想定し、種間で異なるアミノ配列を部分的に移植した変異体を用いての抗腫瘍エピトープの推定を進めることにした。hCD147v1、v2共通に含まれるアミノ酸配列を対象として、カニクイザルとヒトのCD147での配列比較を実施し、種間で異なるアミノ酸領域の9領域に分類し、mu1~mu9とした(図3)。CD147変異体発現ベクター作製と細胞膜上への発現確認用にN末端にFLAG配列を導入したヒトCD147variant2のcDNA配列を人工合成し、pcDNA3.1ベクターに導入したプラスミド、Signal-N-Flag-hCD147v2_pcDNA3.1(Genscript社にて作製)を作製した。さらに同プラスミドのヒトCD147遺伝子にカニクイザルのCD147アミノ酸配列mu1~mu9について、DNA置換によるアミノ酸置換変異として導入したヒト-カニクイザルキメラCD147発現ベクター9種、hCD147-mu1_pcDNA3.1、hCD147-mu2_pcDNA3.1、hCD147-mu3_pcDNA3.1、hCD147-mu4_pcDNA3.1、hCD147-mu5_pcDNA3.1、hCD147-mu6_pcDNA3.1、hCD147-mu7_pcDNA3.1、hCD147-mu8_pcDNA3.1、hCD147-mu9_pcDNA3.1作製した(Genscript社にて作製)。
ヒトCD147、カニクイザルCD147又は、ヒト-カニクイザルキメラCD147発現ベクター9種の発現ベクターをCHO-K1細胞(ATCC、CCL-61)にLipofectamine 2000(サーモサイエンティフィック社、Cat.11668-019)を用いて、導入し、1日後に抗ヒトCD147マウス抗体(LN22R8)、ラット抗ヒトCD147抗体(2P1A6、2P1B7、2P3G8、2P2D10、2P8C12、2P10F2、2P2D6)、10μg/mlで処理し、抗マウスIgG-PE(DAKO社、Cat.R480)、抗ラットIgG-PE(BD社、#550767)を用いて抗CD147抗体のCD147発現CHO-K1細胞への結合を調べた。CD147蛋白質の発現は、市販の抗FLAG抗体(anti-Flag M2, SIGMA社、Cat.F4049-.2MG)を用いて確認した。フローサイトメーター(CantoII、BD Bioscience社)の測定を実施し、表3に結果をまとめた。1次抗体未処理の対照細胞と比較し、10倍以上の蛍光シグナルの増加が認められたサンプルについて、結合陽性(+)と判定した。10倍未満の部分的な蛍光シグナルの増加が認められたサンプルについて、結合弱陽性(±)と判定した。1次抗体未処理の対照細胞と比較し、蛍光シグナルの増加が認められなかったサンプルについて、結合陰性(-)と判定した。
1)-11-1 LN22R8抗体の可変領域をコードするcDNAのヌクレオチド配列の決定
1)-11-1-1 LN22R8抗体生産ハイブリドーマのtotal RNAの調製
LN22R8抗体の可変領域をコードするcDNAを増幅するため、LN22R8抗体産生ハイブリドーマよりTRIzol Reagent(Ambion社)を用いてtotal RNAを調製した。
軽鎖可変領域をコードするcDNAの増幅は、実施例1)-11-1-1で調製したtotal RNAの約1 μgとSMARTer RACE 5’/3’ Kit(Clontech社)を用いて実施した。LN22R8抗体の軽鎖遺伝子の可変領域をコードするcDNAをPCRで増幅するためのプライマーとして、UPM (Universal Primer A Mix:SMARTer RACE 5’/3’ Kitに付属)、及び公知のマウス軽鎖の定常領域の配列から設計したプライマーを用いた。
重鎖可変領域をコードするcDNAの増幅は、実施例1)-11-1-1で調製したtotal RNAの約1μgとSMARTer RACE 5’/3’ Kit(Clontech社)を用いて実施した。LN22R8抗体の重鎖遺伝子の可変領域をコードするcDNAをPCRで増幅するためのプライマーとして、UPM(Universal Primer A Mix:SMARTer RACE 5’/3’ Kitに付属)、及び公知のマウス重鎖の定常領域の配列から設計したプライマーを用いた。
実施例1)-11-1と同様の方法で実施した。ただし、軽鎖遺伝子の可変領域をコードするcDNAをPCRで増幅するためのプライマーとして、UPM(Universal Primer A Mix:SMARTer RACE 5’/3’ Kitに付属)、及び公知のラット軽鎖の定常領域の配列から設計したプライマーを用い、重鎖遺伝子の可変領域をコードするcDNAをPCRで増幅するためのプライマーとして、UPM(Universal Primer A Mix:SMARTer RACE 5’/3’ Kitに付属)、及び公知のラット重鎖の定常領域の配列から設計したプライマーを用いた。
1)-12-1 ヒトキメラ及びヒト化軽鎖発現ベクターpCMA-LKの構築
プラスミドpcDNA3.3-TOPO/LacZ(Invitrogen社)を制限酵素XbaI及びPmeIで消化して得られる約5.4kbのフラグメントと、配列番号27に示すヒト軽鎖シグナル配列及びヒトκ鎖定常領域をコードするDNA配列を含むDNA断片をIn-Fusion HD PCRクローニングキット(Clontech社)を用いて結合して、pcDNA3.3/LKを作製した。
pCMA-LKをXbaI及びPmeIで消化して軽鎖シグナル配列及びヒトκ鎖定常領域を取り除いたDNA断片と、配列番号28で示されるヒト重鎖シグナル配列及びヒトIgG1定常領域のアミノ酸をコードするDNA配列を含むDNA断片をIn-Fusion HD PCRクローニングキット(Clontech社)を用いて結合して、pCMA-G1を構築した。
配列番号29で示されるヒト重鎖シグナル配列及びヒトIgG2定常領域のアミノ酸をコードするDNA配列を含むDNA断片をもちいて、実施例1)-12-2と同様の方法でpCMA-G2を構築した。
実施例1)-11-1-2で得られたLN22R8の軽鎖の可変領域をコードするcDNAをテンプレートとして、In-fusionクローニング用に設計したプライマーでPCRを行うことにより軽鎖の可変領域をコードするcDNAを含むDNA断片を増幅した。pCMA-LKを制限酵素BsiWIで切断した箇所に、In-Fusion HD PCRクローニングキット(Clontech社)を用いて、増幅したDNA断片を挿入することによりヒトキメラLN22R8の軽鎖発現ベクターを構築した。ヒトキメラLN22R8の軽鎖のヌクレオチド配列及び該軽鎖のアミノ酸配列を、配列番号30及び配列番号31にそれぞれ示す。
1)-11-1-3で得られたLN22R8重鎖の可変領域をコードするcDNAをテンプレートとして、In-fusionクローニング用に設計したプライマーでPCRを行うことにより重鎖の可変領域をコードするcDNAを含むDNA断片を増幅した。pCMA-G1を制限酵素BlpIで切断した箇所に、In-Fusion HD PCRクローニングキット(Clontech社)を用いて、増幅したDNA断片を挿入することによりヒトキメラLN22R8のIgG1タイプ重鎖発現ベクターを構築した。ヒトキメラLN22R8のIgG1タイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号32及び配列番号33にそれぞれ示す。
実施例1)-11-1-3で得られたLN22R8重鎖の可変領域をコードするcDNAをテンプレートとして、In-fusionクローニング用に設計したプライマーでPCRを行うことにより重鎖の可変領域をコードするcDNAを含むDNA断片を増幅した。pCMA-G2を制限酵素BlpIで切断した箇所に、In-Fusion HD PCRクローニングキット(Clontech社)を用いて、増幅したDNA断片を挿入することによりヒトキメラLN22R8のIgG2タイプ重鎖発現ベクターを構築した。ヒトキメラLN22R8のIgG2タイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号34及び配列番号35にそれぞれ示す。
配列番号36に示すヒトキメラLN22R8のIgG4Pタイプ重鎖のアミノ酸配列をコードするDNA配列を含むDNA断片を合成した(GENEART社)。合成したDNA断片をもちいて、実施例1)-12-2と同様の方法でヒトキメラLN22R8のIgG4Pタイプ重鎖発現ベクターを構築した。ヒトキメラLN22R8のIgG4Pタイプ重鎖のアミノ酸配列を配列番号37に示す。
1)-13-1 ヒトキメラ及びヒト化IgG1LALAタイプ重鎖発現ベクターpCMA-G1LALAの構築
配列番号38で示されるヒト重鎖シグナル配列及びヒトIgG1LALA定常領域のアミノ酸をコードするDNA配列を含むDNA断片をもちいて、実施例1)-12-2と同様の方法でpCMA-G1LALAを構築した。
配列番号39で示されるヒト重鎖シグナル配列及びヒトIgG4P定常領域のアミノ酸をコードするDNA配列を含むDNA断片をもちいて、実施例1)-12-2と同様の方法でpCMA-G4Pを構築した。
実施例1)-11-2で得られた2P10F2の軽鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-4と同様の方法でヒトキメラ2P10F2の軽鎖発現ベクターを構築した。ヒトキメラ2P10F2の軽鎖のヌクレオチド配列及び該軽鎖のアミノ酸配列を、配列番号40及び配列番号41にそれぞれ示す。
実施例1)-11-2で得られた2P10F2の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、In-fusionクローニング用に設計したプライマーでPCRを行うことにより重鎖の可変領域をコードするcDNAを含むDNA断片を増幅した。pCMA-G1LALAを制限酵素BlpIで切断した箇所に、In-Fusion HD PCRクローニングキット(Clontech社)を用いて、増幅したDNA断片を挿入することによりヒトキメラ2P10F2のIgG1LALAタイプ重鎖発現ベクターを構築した。ヒトキメラ2P10F2のIgG1LALAタイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号42及び配列番号43にそれぞれ示す。
実施例1)-11-2で得られた2P10F2の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-6と同様の方法でヒトキメラ2P10F2のIgG2タイプ重鎖発現ベクターを構築した。ヒトキメラ2P10F2のIgG2タイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号44及び配列番号45にそれぞれ示す。
実施例1)-11-2で得られた2P10F2の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、In-fusionクローニング用に設計したプライマーでPCRを行うことにより重鎖の可変領域をコードするcDNAを含むDNA断片を増幅した。pCMA-G4Pを制限酵素BlpIで切断した箇所に、In-Fusion HD PCRクローニングキット(Clontech社)を用いて、増幅したDNA断片を挿入することによりヒトキメラ2P10F2のIgG4Pタイプ重鎖発現ベクターを構築した。ヒトキメラ2P10F2のIgG4Pタイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号46及び配列番号47にそれぞれ示す。
1)-14-1 LN22R8、2P10F2のヒトキメラ抗体の生産
FreeStyle 293F細胞(Invitrogen社)はマニュアルに従い、継代、培養をおこなった。対数増殖期の1.2×109個のFreeStyle 293F細胞(Invitrogen社)を3L Fernbach Erlenmeyer Flask(CORNING社)に播種し、FreeStyle293 expression medium (Invitrogen社)で希釈して2.0×106細胞/mLに調製した。40mLのOpti-Pro SFM培地(Invitrogen社)に0.24mgの重鎖発現ベクターと0.36mgの軽鎖発現ベクターと1.8mgのPolyethyleneimine(Polyscience #24765)を加えて穏やかに攪拌し、さらに5分間放置した後にFreeStyle 293F細胞に添加した。37℃、8%CO2インキュベーターで4時間、90rpmで振とう培養後に600mLのEX-CELL VPRO培地(SAFC Biosciences社)、18mLのGlutaMAX I(GIBCO社)、及び30mLのYeastolate Ultrafiltrate(GIBCO社)を添加し、37℃、8%CO2インキュベーターで7日間、90rpmで振とう培養して得られた培養上清をDisposable Capsule Filter (Advantec #CCS-045-E1H)でろ過した。
実施例1)-14-1で得られた培養上清から抗体をrProtein Aアフィニティークロマトグラフィーの1段階工程で精製した。培養上清をPBSで平衡化したMabSelectSuReが充填されたカラム(GE Healthcare Bioscience社製)にアプライしたのちに、カラム容量の2倍以上のPBSでカラムを洗浄した。次に2M アルギニン塩酸塩溶液(pH4.0)で溶出し、抗体の含まれる画分を集めた。その画分を透析(Thermo Scientific社、Slide-A-Lyzer Dialysis Cassette)によりHBSor(25mM ヒスチジン/5% ソルビトール、pH6.0)へのバッファー置換を行った。Centrifugal UF Filter Device VIVASPIN20(分画分子量UF10K,Sartorius社)で抗体を濃縮し、IgG濃度を1 mg/mL以上に調製した。最後にMinisart-Plus filter(Sartorius社)でろ過し、精製サンプルとした。
ヒトキメラ抗体のADCC活性について、エフェクター細胞としてヒトの末梢血単核球(PBMC)、ADCC標的細胞としてヒト膵臓株MIA PaCa-2を用いて評価した。放射線同位体51Crで標識したMIA PaCa-2細胞とマウス抗体(LN22R8)、ラット抗体(2P10F2)、又は、ヒトキメラ抗体(LN22R8chIgG1、LN22R8chIgG2、LN22R8chIgG4P、2P10F2chIgG1LALA、2P10F2chIgG4P)を0.5又は、5μg/mlの濃度で4℃30分間処理した後、ヒトの末梢血から分離したPBMCをMIA PaCa-2細胞の20倍の割合で加え、4時間、37度、5%CO2存在下で培養した。上清中に放出された51CrをTopCount NXT v2.53を用いて測定しTotal release値を得た。51Crで標識したMIA PaCa-2細胞をTriton-100で処理して放出された51Crの測定値をMaximamu release値、PBMCを加えない抗体処理細胞から処理して放出された51Crの測定値をSpontaneous release値として、下記の式から、% specific releaseを算出し、図6にまとめた。陰性対照サンプルとして、ヒトIgG(hIgG,ChromPure Human IgG,Jackson ImmunoResearch Laboratories社,Cat.009-000-003)を処理したサンプルについて同様に測定を実施し、併せて示した。測定は3重に実施し、平均値、標準偏差を算出して併せて示した。
ヒトIgG(hIgG)とLN22R8のマウス抗体は、ADCC活性を示さなかったのに対し、LN22R8chIgG1は0.5μg/mlで17.4%、5μg/mlで18.1%とADCC活性を示した。LN22R8chIgG2、LN22R8chIgG4PのADCC活性は、LN22R8chIgG1より低く、5μg/mlでもそれぞれ3.0%、2.2%であった。
抗ヒトCD147抗体による補体依存的な殺細胞活性(CDC活性)を標的細胞としてヒト膵臓株MIA PaCa-2を用いて評価した。補体として市販のウサギ補体(Low Tox-M Rabbit Complement、CEDARLANE LABORATORIES LIMITED、Cat.CL3051)を用いた。抗ヒトCD147抗体として、マウス抗体(LN22R8)、ラット抗体(2P10F2)、又は、ヒトキメラ抗体(LN22R8chIgG1、LN22R8chIgG2、LN22R8chIgG4P、2P10F2chIgG1LALA、2P10F2chIgG4P)を用いた。CDC活性陰性の対照抗体としてヒトIgG(hIgG,ChromPure Human IgG,Jackson ImmunoResearch Laboratories社,Cat.009-000-003)を用いた。上記抗体を0、0.1、1又は、10μg/mlの濃度で1時間、4℃で処理した後、ウサギ補体を終濃度7.5%となるように添加し、37℃、5%CO2存在下で、3時間加温後、生細胞に含まれる細胞内ATPをCellTiter-Glo Lumimescent Cell Viability Assay(Promega社,Cat.G7572)を用いて測定した。CellTiter-Glo Lumimescent Cell Viability Assay を用いて得られる発光シグナルについて、EnVision 2104 Multilabel Reader (Perkin Elmer社)を用いて定量した。測定は3重に実施し、平均値と標準偏差を算出した。無処理の細胞から得られた発光シグナルを100%として、抗体と補体依存的に減少した発光シグナルをCDC活性として、図7にまとめた。
ヒトIgG抗体はマウスのFcγ受容体との相互作用を介して、抗体依存的な単球、マクロファージによる貪食作用(ADCP)を誘導することで、癌細胞への殺細胞活性を示すことが報告されている(Overdijk et al.,Journal of Immunology,1-9,2012)。ヒトキメラ抗体のADCP活性について、エフェクター細胞としてRAW264.7(ATCC,TIB-71)、ADCP標的細胞としてヒト膵臓株PANC-1又はMIA PaCa-2を用いて評価した。PKH67 Green Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling(SIGMA,Cat.MINI67-1KIT)で標識したADCP標的細胞とヒトキメラ抗体(LN22R8chIgG1、LN22R8chIgG2、LN22R8chIgG4P)を20μg/mlの濃度で4℃、1時間処理した後、PKH26 Red Fluorescent Cell Linker Lit for General Cell Membrane Labeling(SIGMA,Cat.PKH26GL-1KT)で標識したRAW264.7細胞をADCP標的細胞の5倍添加し、3時間、37℃、5% CO2存在下で加温した。フローサイトメーター(BD社、CantoII)を用いて、貪食作用によりPKH67シグナル陽性に移行したPKH26陽性細胞の割合を測定した。陰性対照サンプルとして、ヒトIgG(hIgG, ChromPure Human IgG,Jackson ImmunoResearch Laboratories社,Cat.009-000-003)を処理したサンプルについて同様に測定を実施した。測定は3重に実施し、平均値、標準偏差を算出して図8(a)PANC-1の結果を、図8(b)MIA PaCa-2の結果をそれぞれ示した。
5×106細胞のヒト膵臓株MIA PaCa-2を50%GFR-Matrigel(Corning社、Cat.354230)を含むPBSで懸濁し、4~5週齢雌のNOD-scidマウス(NOD.CB17-Prkdc<scid>/J、日本チャールス・リバーより購入)の腋窩部皮下に移植した。腫瘍体積をもとに移植の5~7日後に群分けを実施し、抗ヒトCD147抗体LN22R8のマウス抗体(LN22R8)、ヒトキメラ抗体3種(LN22R8chIgG1、LN22R8chIgG2、LN22R8chIgG4P)を1mg/kg、3mg/kg、10mg/kgで担癌マウスの腹腔内に投与した(n=5)。抗ヒトCD147抗体2P10F2のラット抗体(2P10F2)、ヒトキメラ抗体2種(2P10F2chIgG2,2P10F2chIgG4P)を10mg/kgで担癌マウスの腹腔内に投与した(n=5~6)。移植腫瘍の長径及び短径を週2回、電子デジタルノギス(株式会社ミツトヨ製)を用いて測定し、以下に示す計算式により腫瘍体積を算出した。
結果を図9-1(a)~(d)、図9-2(e)~(g)に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。
NOG(NOD/Shi-scid, IL-2Rγnull)マウスでは、マウスT細胞、B細胞を欠失しているNOD-scidマウスにサイトカインレセプター共通ドメインであるIL-2レセプターγ鎖ノックアウトを掛け合わせることで、マウスT、B細胞に加え、NK細胞、補体活性を欠失しており、マクロファージや樹上細胞の機能低下がみられ、極めて重度な免疫不全状態にある(Ito,Blood,3175-3182,2002)。これらマウス免疫系の重度な不全状態で、CD147抗体の抗腫瘍効果が影響を受けるかMIA PaCa-2の皮下移植モデルで検証した。
結果を図10に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。
実施例1で得られた強い抗腫瘍効果を示す抗ヒトCD147抗体は、マウス、ラット、カニクイザルのCD147に交差性を示さなかった。実施例1で得られた抗体を用いてカニクイザルCD147交差性を示すCD147抗体の取得を試みた。
免疫にはWKY/Izmラットの雌(日本エスエルシー社)を使用した。Recombinant Human BSG, His tagged(CREATIVE BIOMART社製)抗原蛋白とFreund‘s Comple te Adjuvant(和光純薬社製)を混合したものを尾根部に投与したラットのリンパ節及び脾臓を採取しハイブリドーマ作製に用いた。
リンパ節細胞若しくは脾臓細胞とマウスミエローマSP2/0-ag14細胞(ATCC,No.CRL-1581)とをLF301 Cell Fusion Unit(BEX社) を用いて電気細胞融合し、ClonaCell-HY Selection Medium D(StemCell Technologies社)に希釈して培養した。出現したハイブリドーマコロニーを回収することでモノクローンハイブリドーマを作製した。回収された各ハイブリドーマコロニーを培養し、得られたハイブリドーマ培養上清を用いて抗CD147抗体産生ハイブリドーマのスクリーニングを行った。
ヒト癌細胞に結合し、ヒトCD147、カニクイザルCD147への結合性を示す抗体産生ハイブリドーマを選択するためにフローサイトメーターを用いた抗体結合性スクリーニングを実施した。ヒト癌細胞として、CD147陽性のヒト膵臓株MIA PaCa-2を用いた。実施例1)-8と同様にヒト、又は、カニクイザルのCD147を発現したCHO-K1細胞(CHO-K1-hCD147v2、CHO-K1-cynoCD147)をヒト、又はカニクイザルCD147への結合性確認に用いた。MIA PaCa-2、CHO-K1-hCD147v2、CHO-K1-cynoCD147の懸濁液にハイブリドーマ培養上清を等量添加し、4℃で1時間以上反応させた後、5%FBSを含むPBSで細胞を洗浄し、抗ラットIgG-PE(BD Biosciences社、Cat.550767)を用いて各抗体の細胞への結合を蛍光検出可能にした。フローサイトメーター(CantoII、BD Bioscience社)を用いて細胞の蛍光シグナルの測定を実施し、陰性対照サンプル(ハイブリドーマ培養液を加えていない細胞)に対する蛍光シグナルの比を算出し、表2に結果の一部をまとめた。
カニクイザル交差性を示す抗ヒトCD147モノクローナル抗体は、ハイブリドーマ培養上清から精製した。まず、rat_CD147_#131の抗体産生ハイブリドーマをClonaCell-HY Selection Med ium Eで十分量まで増殖させた後、Ultra Low IgG FBS(Life Technologies社)を20%添加したHybridoma SFM(Life Technologies社)に培地交換し、7日間培養した。本培養上清を回収し0.45μmのフィルターを通して滅菌した。
CL-1000フラスコ(日本ベクトン・ディッキンソン株式会社)を用いて、rat_CD147_#84、rat_CD147_#101、rat_CD147_#110ハイブリドーマを培養し、モノクローナル抗体を含むハイブリドーマ培養上清を調製した。
実施例2)-4及び実施例2)-5で作製した培養上清から実施例1)-6と同様の方法で抗体を精製した。
5×106細胞のヒト膵臓株MIA PaCa-2を50%GFR-Matrigel(Corning社、Cat. 354230)を含むPBSで懸濁し、5週齢雌のNOD-scidマウス(NOD.CB17-Prkdc<scid>/J、日本チャールス・リバーより購入)の腋窩部皮下に移植した。腫瘍体積をもとに移植の6~8日後に群分けを実施し、カニクイザル交差性抗CD147ラット抗体#84、#101又は#110を移植後、8日後、15日後に10mg/kgで担癌マウスの腹腔内に投与した(n=5)。対照群のマウスには、PBSを同様に腹腔内に投与した。カニクイザル交差性抗CD147ラット抗体#131を移植後、6日後に10mg/kgで担癌マウスの腹腔内に投与した(n=5)。移植腫瘍の長径及び短径を週2回、電子デジタルノギス(株式会社ミツトヨ製)を用いて測定し、以下に示す計算式により腫瘍体積を算出した。
結果を図11(a)~(d)に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。r#84は、移植28日後における腫瘍増殖抑制率が、10mg/kg投与群で95%であった(図11(a))。r#101は、移植15日後における腫瘍増殖抑制率が10mg/kg投与群で37%であったが、移植28日後には腫瘍の再増殖が見られた(図11(b))。r#110は、移植15日後における腫瘍増殖抑制率が10mg/kg投与群で51%であったが、移植28日後には腫瘍の再増殖が見られた(図11(c))。r#131は、移植16日後における腫瘍増殖抑制率は、10mg/kg投与群で50%であった(図11(d))。MIA PaCa-2の腫瘍増殖を強く阻害するラット抗体、r#84、部分的な阻害をするr#101、r#110、r#131が得られた。
サル交差性ラットCD147抗体のエピトープ解析を目的として、2P10F2chIgG4PのCD147リコンビナント蛋白質への結合性を阻害するか競合ELISAにより調べた。ヒトCD147-Fc融合蛋白質(Sino Biological Inc.,10186-H02H)をPBSで溶解し、20μg/mlに調製し、96ウェルプレート(Thermofisher社,Cat.43454)に50μl加え4℃で保管した。蛋白溶液を除き、300μlの1%BSA含有PBSを加えて、室温で1時間加温した。1%BSA含有PBS で希釈した25μlの20あるいは60μg/mlのCD147ラット抗体r#84、r#101、r#110、r#131又は2P10F2、又は1%BSA含有PBSを競合抗体として96ウェルプレートに加え、室温で2時間加温した。1%BSA含有PBS で希釈した20 ng/mlの 2P10F2chIgG4Pを25μl、96ウェルプレートに加え、室温で2時間加温した。0.05% Tween 20(BIO RAD,Cat.170-6531)を含むPBSで2回、96ウェルを洗浄した。1%BSA含有PBS で2000倍に希釈したMouse monoclonal HP6025 Anti-Human IgG4 Fc(HRP)(abcam社,Cat.ab99823)を50μl、96ウェルプレートに加え、室温で1時間加温した。0.05% Tween 20(BIO RAD,Cat.170-6531)を含むPBSで3回、96ウェルを洗浄した。50μlのSuper AquaBlue ELISA Substrate (eBioscience社,00-4203-58)を96ウェルプレートに加え、室温で20分間加温した。EnVision 2104 Multilabel Reader(Perkin Elmer社)で、96ウェルプレートの405nmの吸光度を測定した。競合抗体を含まないウェルでの測定値を対照として、競合抗体によって低下した吸光度を%で算出し図12に示した。測定は3ウェルで実施し、平均値を示した。
3)-1 ラット抗CD147抗体の可変領域をコードするcDNAのクローニングヌクレオチド配列の決定
3)-1-1 rat_CD147_#84抗体の可変領域をコードするcDNAのヌクレオチド配列の決定
実施例1)-11-2と同様の方法で実施した。決定されたrat_CD147_#84抗体の軽鎖の可変領域をコードするcDNAのヌクレオチド配列を配列番号48に示し、アミノ酸配列を配列番号49に示す。重鎖の可変領域をコードするcDNAのヌクレオチド配列を配列番号50に示し、アミノ酸配列を配列番号51に示す。rat_CD147_#84抗体の軽鎖可変領域のCDRL1、CDRL2及びCDRL3を、それぞれ、配列番号52、53及び54に示す。rat_CD147_#84抗体の重鎖可変領域のCDRH1、CDRH2及びCDRH3を、それぞれ、配列番号55、56及び57に示す。
実施例1)-11-2と同様の方法で実施した。決定されたrat_CD147_#101抗体の軽鎖の可変領域をコードするcDNAのヌクレオチド配列を配列番号58に示し、アミノ酸配列を配列番号59に示す。重鎖の可変領域をコードするcDNAのヌクレオチド配列を配列番号60に示し、アミノ酸配列を配列番号61に示す。rat_CD147_#101抗体の軽鎖可変領域のCDRL1、CDRL2及びCDRL3を、それぞれ、配列番号62、63及び64に示す。rat_CD147_#101抗体の重鎖可変領域のCDRH1、CDRH2及びCDRH3を、それぞれ、配列番号65、66及び67に示す。
実施例1)-11-2と同様の方法で実施した。決定されたrat_CD147_#110抗体の軽鎖の可変領域をコードするcDNAのヌクレオチド配列を配列番号68に示し、アミノ酸配列を配列番号69に示す。重鎖の可変領域をコードするcDNAのヌクレオチド配列を配列番号70に示し、アミノ酸配列を配列番号71に示す。rat_CD147_#110抗体の軽鎖可変領域のCDRL1、CDRL2及びCDRL3を、それぞれ、配列番号72、73及び74に示す。rat_CD147_#110抗体の重鎖可変領域のCDRH1、CDRH2及びCDRH3を、それぞれ、配列番号75、76及び77に示す。
実施例1)-11-2と同様の方法で実施した。決定されたrat_CD147_#131抗体の軽鎖の可変領域をコードするcDNAのヌクレオチド配列を配列番号78に示し、アミノ酸配列を配列番号79に示す。重鎖の可変領域をコードするcDNAのヌクレオチド配列を配列番号80に示し、アミノ酸配列を配列番号81に示す。rat_CD147_#131抗体の軽鎖可変領域のCDRL1、CDRL2及びCDRL3を、それぞれ、配列番号82、83及び84に示す。rat_CD147_#131抗体の重鎖可変領域のCDRH1、CDRH2及びCDRH3を、それぞれ、配列番号85、86及び87に示す。
3)-2-1 rat_CD147_#84のヒトキメラ抗体発現ベクターの作製
3)-2-1-1 ヒトキメラ及びヒト化IgG4PFALAタイプ重鎖発現ベクターpCMA-G4PFALAの構築
配列番号88で示されるヒト重鎖シグナル配列及びヒトIgG4PFALA定常領域のアミノ酸をコードするDNA配列を含むDNA断片をもちいて、実施例1)-12-2と同様の方法でpCMA-G4PFALAを構築した。
実施例3)-1-1で得られたrat_CD147_#84の軽鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-4と同様の方法でヒトキメラrat_CD147_#84の軽鎖発現ベクターを構築した。ヒトキメラrat_CD147_#84の軽鎖のヌクレオチド配列及び該軽鎖のアミノ酸配列を、配列番号89及び配列番号90にそれぞれ示す。
実施例3)-1-1で得られたrat_CD147_#84の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-5と同様の方法でヒトキメラrat_CD147_#84のIgG1タイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#84のIgG1タイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号91及び配列番号92にそれぞれ示す。
実施例3)-1-1で得られたrat_CD147_#84の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-6と同様の方法でヒトキメラrat_CD147_#84のIgG2タイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#84のIgG2タイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号93及び配列番号94にそれぞれ示す。
実施例3)-1-1で得られたrat_CD147_#84の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-13-6と同様の方法でヒトキメラrat_CD147_#84のIgG4Pタイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#84のIgG4Pタイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号95及び配列番号96にそれぞれ示す。
実施例3)-1-1で得られたrat_CD147_#84の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-13-4と同様の方法でヒトキメラrat_CD147_#84のIgG1LALAタイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#84のIgG1LALAタイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号97及び配列番号98にそれぞれ示す。
実施例3)-1-1で得られたrat_CD147_#84の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、In-fusionクローニング用に設計したプライマーでPCRを行うことにより重鎖の可変領域をコードするcDNAを含むDNA断片を増幅した。pCMA-G4PFALAを制限酵素BlpIで切断した箇所に、In-Fusion HD PCRクローニングキット(Clontech社)を用いて、増幅したDNA断片を挿入することによりヒトキメラrat_CD147_#84のIgG4PFALAタイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#84のIgG4PFALAタイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号99及び配列番号100にそれぞれ示す。
3)-2-2-1 ヒトキメラrat_CD147_#101の軽鎖発現ベクターの構築
実施例3)-1-2で得られたrat_CD147_#101の軽鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-4と同様の方法でヒトキメラrat_CD147_#101の軽鎖発現ベクターを構築した。ヒトキメラrat_CD147_#101の軽鎖のヌクレオチド配列及び該軽鎖のアミノ酸配列を、配列番号101及び配列番号102にそれぞれ示す。
実施例3)-1-2で得られたrat_CD147_#101の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-6と同様の方法でヒトキメラrat_CD147_#101のIgG2タイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#101のIgG2タイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号103及び配列番号104にそれぞれ示す。
実施例3)-1-2で得られたrat_CD147_#101の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-13-6と同様の方法でヒトキメラrat_CD147_#101のIgG4Pタイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#101のIgG4Pタイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号105及び配列番号106にそれぞれ示す。
実施例3)-1-2で得られたrat_CD147_#101の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例3)-2-1-7と同様の方法でヒトキメラrat_CD147_#101のIgG4PFALAタイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#101のIgG4PFALAタイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号107及び配列番号108にそれぞれ示す。
3)-2-3-1 ヒトキメラrat_CD147_#110の軽鎖発現ベクターの構築
実施例3)-1-3で得られたrat_CD147_#110の軽鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-4と同様の方法でヒトキメラrat_CD147_#110の軽鎖発現ベクターを構築した。ヒトキメラrat_CD147_#110の軽鎖のヌクレオチド配列及び該軽鎖のアミノ酸配列を、配列番号109及び配列番号110にそれぞれ示す。
実施例3)-1-3で得られたrat_CD147_#110の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-6と同様の方法でヒトキメラrat_CD147_#110のIgG2タイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#110のIgG2タイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号111及び配列番号112にそれぞれ示す。
実施例3)-1-3で得られたrat_CD147_#110の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-13-6と同様の方法でヒトキメラrat_CD147_#110のIgG4Pタイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#110のIgG4Pタイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号113及び配列番号114にそれぞれ示す。
実施例3)-1-3で得られたrat_CD147_#110の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例3)-2-1-7と同様の方法でヒトキメラrat_CD147_#110のIgG4PFALAタイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#110のIgG4PFALAタイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号115及び配列番号116にそれぞれ示す。
3)-2-4-1 ヒトキメラrat_CD147_#131の軽鎖発現ベクターの構築
実施例3)-1-4で得られたrat_CD147_#131の軽鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-4と同様の方法でヒトキメラrat_CD147_#131の軽鎖発現ベクターを構築した。ヒトキメラrat_CD147_#131の軽鎖のヌクレオチド配列及び該軽鎖のアミノ酸配列を、配列番号117及び配列番号118にそれぞれ示す。
実施例3)-1-4で得られたrat_CD147_#131の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-12-6と同様の方法でヒトキメラrat_CD147_#131のIgG2タイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#131のIgG2タイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号119及び配列番号120にそれぞれ示す。
実施例3)-1-4で得られたrat_CD147_#131の重鎖の可変領域をコードするcDNAをテンプレートとしてもちいて、実施例1)-13-6と同様の方法でヒトキメラrat_CD147_#131のIgG4Pタイプ重鎖発現ベクターを構築した。ヒトキメラrat_CD147_#131のIgG4Pタイプ重鎖のヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号121及び配列番号122にそれぞれ示す。
3)-3-1 サル交差性ラット抗体のヒトキメラ抗体の生産
実施例1)-14-1と同様の方法で生産した。
実施例3)-3-1で得られた培養上清から、実施例1)-14-2と同様の方法で精製した。
5×106細胞のヒト膵臓株MIA PaCa-2を50%GFR-Matrigel(Corning社、Cat.354230)を含むPBSで懸濁し、5~6週齢雌のNOD-scidマウス(NOD.CB17-Prkdc<scid>/J、日本チャールス・リバーより購入)の腋窩部皮下に移植した。腫瘍体積をもとに移植の5~6日後に群分けを実施し、カニクイザル交差性抗CD147ヒトキメラ抗体(#84chIgG1、#84chIgG1LALA、#84chIgG2、#84chIgG4P、#84chIgG4PFALA)を群分け後当日に1、3、又は10 mg/kgで担癌マウスの腹腔内に投与した(n=5)。カニクイザル交差性抗CD147ヒトキメラ抗体(#101chIgG2、#101chIgG4P、#101chIgG4PFALA、#110chIgG2、#110chIgG4P、#110chIgG4PFALA、#131chIgG2、#131chIgG4P)を群分け後当日に3、又は10mg/kgで担癌マウスの腹腔内に投与した(n=5)。移植腫瘍の長径及び短径を週2回、電子デジタルノギス(株式会社ミツトヨ製)を用いて測定し、以下に示す計算式により腫瘍体積を算出した。
結果を図13-1(a)~(d)、図13-2(e)~(h)、図13-3(i)~(l)、図13-4(m)及び(n)に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。
実施例3)-3-1で作製した#84chIgG1、#84chIgG2、#84chIgG4P、#84chIgG1LALA、#84chIgG4PFALA、#101chIgG4P、#110chIgG4PのヒトCD147に対する解離定数測定は、Biacore T200(GE Healthcare Bioscience社製)を使用し、Human Antibody Capture Kit(GE Healthcare Bioscience社製) を用いて固定化したAnti-Human IgG(Fc)antibodyに抗体をリガンドとして捕捉(キャプチャー)し、抗原をアナライトとして測定するキャプチャー法にて行った。ランニングバッファーとしてHBS-EP+(GE Healthcare Bioscience社製)、センサーチップとしてCM5(GE Healthcare Bioscience社製)を用いた。チップ上に1μg/mL又は2μg/mLのヒトキメラ抗体を10μL/分で60秒間添加した後、抗原としてCD147蛋白質の希釈系列溶液(#131chIgG4Pに対して0.25~4μg/mL、#84chIgG1、#84chIgG2、#84chIgG4P、#84chIgG1LALA、#84chIgG4PFALA、#101chIgG4P、#110chIgG4Pに対して0.5~8μg/mL)を流速30μL/分で120秒間添加し、引き続き120秒間、300秒間又は600秒間の解離相をモニターした。ここで、CD147蛋白質は、大腸菌で発現させ、Ni affinity, SECの2 steps精製後、タグ切断精製したものを使用した。再生溶液として、3M magnesium chloride(GE Healthcare Bioscience社製)を流速20μL/分で30秒間添加した。データの解析には1:1結合モデルを用いて、結合速度定数ka、解離速度定数kd及び解離定数(KD;KD=kd/ka)を算出した。結果を表5に示す。
6)-1 ヒト化抗体の設計
6)-1-1 可変領域の分子モデリング
ホモロジーモデリングとして公知の方法(Methods in Enzymology,203,121-153,(1991))を利用した。市販のタンパク質立体構造解析プログラムDiscovery Studio(ダッソー・システムズ社製)を用いて、可変領域に対して高い配列同一性を有するProtein Data Bank(Nuc.Acids Res.35,D301-D303(2007))に登録されている構造を検索した。ヒットした重鎖、軽鎖及び重鎖軽鎖インターフェース構造を鋳型として、三次元モデルが作成された。
CDRグラフティング(Proc.Natl.Acad.Sci.USA 86,10029-10033(1989))によりヒト化した。rat_CD147_#84のフレームワーク領域は、KABAT et al.(Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service National Institutes of Health,Bethesda,MD.(1991))において既定されるヒトkappa鎖サブグループ1のコンセンサス配列と、ヒトgamma鎖サブグループ3のコンセンサス配列に高い相同性を有することから、それらがrat_CD147_#84の軽鎖と重鎖のアクセプタとしてそれぞれ選択された。rat_CD147_#101のフレームワーク領域は、KABAT et al.において既定されるヒトkappa鎖サブグループ1のコンセンサス配列と、ヒトgamma鎖サブグループ3及びIMGT(THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM(登録商標))において規定されるヒトgamma鎖のIGHV3-30*05とIGHJ3*01に、高い相同性を有することから、それらがrat_CD147_#101の軽鎖と重鎖のアクセプタとしてそれぞれ選択された。rat_CD147_#110のフレームワーク領域は、IMGTにおいて規定されるヒトkappa鎖のIGKV1-39*01とIGKJ4*01と、ヒトgamma鎖のIGHV1-2*02とIGHJ6*01に、高い相同性を有することから、それらがrat_CD147_#110の軽鎖と重鎖のアクセプタとしてそれぞれ選択された。rat_CD147_#131のフレームワーク領域は、IMGTにおいて規定されるヒトkappa鎖のIGKV1-39*01とIGKJ2*01と、ヒトgamma鎖のIGHV3-30*05とIGHJ6*01に、高い相同性を有することから、rat_CD147_#131の軽鎖と重鎖のアクセプタとしてそれぞれ選択された。アクセプタ上に移入すべきドナー残基は、Queen et al.(Proc.Natl.Acad.Sci.USA 86,10029-10033(1989))によって与えられる基準などを参考に、三次元モデルを分析することで選択された。
rat_CD147_#84重鎖の可変領域に対し、図14(a)に示すアクセプタのアミノ酸残基を移入することで、ヒト化抗体重鎖#84H1hの可変領域が設計された。
rat_CD147_#84軽鎖の可変領域に対し、図14(b)に示すアクセプタのアミノ酸残基を移入することで、ヒト化抗体軽鎖#84L2hの可変領域が設計された。
上記より設計された重鎖#84H1hIgG2と軽鎖#84L2hを組み合わせ、ヒト化抗体#84H1L2hIgG2を設計した。また、重鎖#84H1hIgG4Pと軽鎖#84L2hを組み合わせ、ヒト化抗体#84H1L2hIgG4Pを設計した。
rat_CD147_#101重鎖の可変領域に対し、図15(a)に示すアクセプタのアミノ酸残基を移入することで、ヒト化抗体重鎖#101H1hの可変領域が設計された。
設計された可変領域に、ヒトのIgG2及びIgG4Pのgamma鎖定常領域を接続させたヒト化抗体重鎖が設計され、それぞれ#101H1hIgG2、#101H1hIgG4Pと命名した。#101H1hIgG2の全長アミノ酸配列を配列番号129に記載する。配列番号129のアミノ酸配列をコードするヌクレオチド配列を配列番号130に記載する。#101H1hIgG4Pの全長アミノ酸配列を配列番号131に記載する。配列番号131のアミノ酸配列をコードするヌクレオチド配列を配列番号132に記載する。
rat_CD147_#101軽鎖の可変領域に対し、図15(b)に示すアクセプタのアミノ酸残基を移入することで、ヒト化抗体軽鎖#101L2hの可変領域が設計された。
設計された可変領域に、ヒトのκ鎖定常領域を接続させたヒト化抗体軽鎖が設計され、#101L2hと命名した。#101L2hの軽鎖全長アミノ酸配列を配列番号133に記載する。配列番号133のアミノ酸配列をコードするヌクレオチド配列を配列番号134に記載する。
上記より設計された重鎖#101H1hIgG2と軽鎖#101L2hを組み合わせ、ヒト化抗体#101H1L2hIgG2を設計した。また、重鎖#101H1hIgG4Pと軽鎖#101L2hを組み合わせ、ヒト化抗体#101H1L2hIgG4Pを設計した。
rat_CD147_#110重鎖の可変領域に対し、図16(a)、(b)に示すアクセプタのアミノ酸残基を移入することで、ヒト化抗体重鎖#110H1h、#110H13hの可変領域が設計された。
rat_CD147_#110軽鎖の可変領域に対し、図16(c)、(d)、(e)に示すアクセプタのアミノ酸残基を移入することで、ヒト化抗体軽鎖#110L4h、#110L2h、#110L12hの可変領域が設計された。
上記より設計された重鎖#110H1hIgG4Pと軽鎖#110L4hを組み合わせ、ヒト化抗体#110H1L4hIgG4Pを設計した。また、重鎖#110H13hIgG4Pと軽鎖#110L2hを組み合わせ、ヒト化抗体#110H13L2hIgG4Pを設計し、重鎖#110H13hIgG4Pと軽鎖#110L12hを組み合わせ、ヒト化抗体#110H13L12hIgG4Pを設計した。
rat_CD147_#131重鎖の可変領域に対し、図17(a)に示すアクセプタのアミノ酸残基を移入することで、ヒト化抗体重鎖#131H2hの可変領域が設計された。
rat_CD147_#131軽鎖の可変領域に対し、図17(b)に示すアクセプタのアミノ酸残基を移入することで、ヒト化抗体軽鎖#131L2hの可変領域が設計された。
上記より設計された重鎖#131H2hIgG2と軽鎖#131L2hを組み合わせ、ヒト化抗体#131H2L2hIgG2を設計した。
6)-2-1 #84L2h発現ベクターの構築
配列番号128に示す#84L2hのヌクレオチド配列のヌクレオチド番号37乃至402に示されるDNA断片を合成した(GENEART社)。In-Fusion HD PCRクローニングキット(Clontech社)を用いて、pCMA-LKを制限酵素BsiWIで切断した箇所に合成したDNA断片を挿入することにより#84L2h発現ベクターを構築した。
配列番号134に示す#101L2hのヌクレオチド配列のヌクレオチド番号37乃至402に示されるDNA断片を合成した(GENEART社)。実施例6)-2-1と同様の方法で#101L2h発現ベクターを構築した。
配列番号138、配列番号150及び配列番号152に示す#110L4h、#110L2h及び#110L12hのヌクレオチド配列のヌクレオチド番号37乃至402に示されるDNA断片を合成した(GENEART社)。実施例6)-2-1と同様の方法で#110L4h、#110L2h及び#110L12h発現ベクターを構築した。
配列番号142に示す#131L2hのヌクレオチド配列のヌクレオチド番号37乃至402に示されるDNA断片を合成した(GENEART社)。実施例6)-2-1と同様の方法で#131L2h発現ベクターを構築した。
6)-3-1 #84H1hIgG2発現ベクターの構築
配列番号124に示す#84H1hIgG2のヌクレオチド配列のヌクレオチド番号36乃至437に示されるDNA断片を合成した(GENEART社)。In-Fusion HD PCRクローニングキット(Clontech社)を用いて、pCMA-G2を制限酵素BlpIで切断した箇所に合成したDNA断片を挿入することにより#84H1hIgG2発現ベクターを構築した。
配列番号126に示す#84H1hIgG4Pのヌクレオチド配列のヌクレオチド番号36乃至437に示されるDNA断片を合成した(GENEART社)。In-Fusion HD PCRクローニングキット(Clontech社)を用いて、pCMA-G4Pを制限酵素BlpIで切断した箇所に合成したDNA断片を挿入することにより#84H1hIgG4P発現ベクターを構築した。
配列番号130に示す#101H1hIgG2のヌクレオチド配列のヌクレオチド番号36乃至428に示されるDNA断片を合成した(GENEART社)。実施例6)-3-1と同様の方法で#101H1hIgG2発現ベクターを構築した。
配列番号132に示す#101H1hIgG4Pのヌクレオチド配列のヌクレオチド番号36乃至428に示されるDNA断片を合成した(GENEART社)。実施例6)-3-2と同様の方法で#101H1hIgG4P発現ベクターを構築した。
配列番号136及び配列番号148に示す#110H1hIgG4P及び#110H13hIgG4Pのヌクレオチド配列のヌクレオチド番号36乃至425に示されるDNA断片を合成した(GENEART社)。実施例6)-3-2と同様の方法で#110H1hIgG4P及び#110H13hIgG4P発現ベクターを構築した。
配列番号140に示す#131H2hIgG2のヌクレオチド配列のヌクレオチド番号36乃至431に示されるDNA断片を合成した(GENEART社)。実施例6)-3-1と同様の方法で#131H2hIgG2発現ベクターを構築した。
6)-4-1 ヒト化抗体の生産
実施例1)-14-1と同様の方法で生産した。実施例6)-1-5、実施例6)-1-8、実施例6)-1-11、及び実施例6)-1-14に示したH鎖とL鎖の組み合わせに対応したH鎖発現ベクターとL鎖発現ベクターの組み合わせで、各種ヒト化抗体を取得した。
実施例6)-4-1で得られた培養上清から抗体をrProtein Aアフィニティークロマトグラフィーの1段階工程で精製した。培養上清をPBSで平衡化したMabSelectSuReが充填されたカラム(GE Healthcare Bioscience社製)にアプライしたのちに、カラム容量の2倍以上のPBSでカラムを洗浄した。次に2Mアルギニン塩酸塩溶液(pH4.0)で溶出し、抗体の含まれる画分を集めた。その画分を透析(Thermo Scientific社、Slide-A-Lyzer Dialysis Cassette)によりPBSへのバッファー置換を行った。Centrifugal UF Filter Device VIVASPIN20(分画分子量UF10K,Sartorius社)で抗体を濃縮し、IgG濃度を1mg/mL以上に調製した。最後にMinisart-Plus filter(Sartorius社)でろ過し、精製サンプルとした。
実施例6)-4-1で得られた培養上清をrProtein Aアフィニティークロマトグラフィーとセラミックハイドロキシアパタイトの2段階工程で精製した。培養上清をPBSで平衡化したMabSelectSuReが充填されたカラム(GE Healthcare Bioscience社製)にアプライした後に、カラム容量の2倍以上のPBSでカラムを洗浄した。次に2 Mアルギニン塩酸塩溶液(pH4.0)で抗体を溶出した。抗体の含まれる画分を透析(Thermo Scientific社、Slide-A-Lyzer Dialysis Cassette)によりPBSへのバッファー置換を行い、5 mMリン酸ナトリウム/50 mM MES/pH7.0のバッファーで5倍希釈した後に、5 mM NaPi/50 mM MES/30 mM NaCl/pH7.0のバッファーで平衡化したセラミックハイドロキシアパタイトカラム(日本バイオラッド、Bio-Scale CHT Type―1 Hydroxyapatite Column)にアプライした。塩化ナトリウムによる直線的濃度勾配溶出を実施し、抗体の含まれる画分を集めた。その画分を透析(Thermo Scientific社、Slide-A-Lyzer Dialysis Cassette)によりHBSor(25 mM ヒスチジン/5% ソルビトール、pH6.0)へのバッファー置換を行った。Centrifugal UF Filter Device VIVASPIN20(分画分子量UF10K,Sartorius社)にて抗体を濃縮し、IgG濃度を25 mg/mLに調製した。最後にMinisart-Plus filter(Sartorius社)でろ過し、精製サンプルとした。
7)-1 ヒト化抗体のCD147抗体結合性評価
実施例6)-4-3で作製したヒト化抗ヒトCD147抗体#84H1L2hIgG2、#84H1L2hIgG4P、#101H1L2hIgG2、#101H1L2hIgG4P、#110H1L4hIgG4P及び#131H2L2hIgG2とヒトCD147との解離定数測定は、Biacore T200(GE Healthcare Bioscience社製)を使用し、Human Antibody Capture Kit(GE Healthcare Bioscience社製)を用いて固定化したAnti-Human IgG(Fc) antibodyに抗体をリガンドとして捕捉(キャプチャー)し、抗原をアナライトとして測定するキャプチャー法にて行った。ランニングバッファーとしてHBS-EP+(GE Healthcare Bioscience社製)、センサーチップとしてCM5(GE Healthcare Bioscience社製)を用いた。チップ上に1μg/mLのヒト化抗体を10μL/分で60秒間添加した後、実施例5)で用いた抗原の希釈系列溶液(#101H1L2hIgG2及び#101H1L2hIgG4Pに対して0.0625~1μg/mL、#84H1L2hIgG2、#84H1L2hIgG4P、#110H1L4hIgG4P、#110H13L2hIgG4P、#110H13L12hIgG4P及び#131H2L2hIgG2に対して0.25~4μg/mL)を流速30μL/分で120秒間添加し、引き続き300秒間の解離相をモニターした。再生溶液として、3M magnesium chloride(GE Healthcare Bioscience社製)を流速20μL/分で30秒間添加した。データの解析には1:1結合モデルを用いて、結合速度定数ka、解離速度定数kd及び解離定数(KD;KD=kd/ka)を算出した。
5×106細胞のヒト膵臓株MIA PaCa-2を50%GFR-Matrigel(Corning社、Cat. 354230)を含むPBSで懸濁し、4週齢雌のNOD-scidマウス(NOD.CB17-Prkdc<scid>/J、日本チャールス・リバーより購入)の腋窩部皮下に移植した。腫瘍体積をもとに群分けをし、移植の7日後に実施例6)-4-2で作製したヒト化CD147抗体(#84H1L2hIgG2、#84H1L2Ig4P、#101H1L2hIg2、#101H1L2hIg4P、#110H1L4hIg4P、#131H2L2hIg2)を3mg/kg 、10 mg/kgで担癌マウスの腹腔内に投与した(n=5)。対照薬として膵臓癌の治療薬であるゲムシタビン(日本イーライリリー社より購入)を移植の7、14日後に400 mg/kgで担癌マウスの腹腔内に投与した(n=5)。移植腫瘍の長径及び短径を週2回、電子デジタルノギス(株式会社ミツトヨ製)を用いて測定し、以下に示す計算式により腫瘍体積を算出した。
8)-1 膵臓癌細胞PANC-1における抗ヒトCD147ヒトキメラ抗体によるp38MAPKリン酸化の誘導
CD147は活性化によってp38MAPKのリン酸化を促すことが報告されている(Lim et al.,FEBS Letters、88-92,1998)(Li et.,al.J.Hepatology,1378-1389,2015)。抗腫瘍効果を示す抗CD147が、p38MAPKのシグナルに及ぼす影響を調べるために、CD147ヒトキメラ抗体LN22R8chIgG4Pを10 μg/mlで15分間処理したPANC-1細胞について、p38MAPKリン酸化をSimple Western assays法(プロテインシンプル ジャパン株式会社、Wes)を用いて評価した。対照サンプルとして、PANC-1細胞をヒトIgG(Jackson ImmunoResearch社、009-000-003)10 μg/mlを同様に処理して解析に供した。p38MAPKの検出には、p38 MAPK rabbit mAb(Cell signaling technology社、Cat.#9212)を用いた。リン酸化p38MAPKの検出には、P-p38 MAPK(T180/Y182)(D3F9)XP rabbit mAb(Cell signaling technology社、#4511S)を用いた。検出されたp38MAPKシグナル対する、リン酸化p38MAPKシグナルの比を図19に示した。抗体処理サンプルは2重に実施し、平均値を図に示した。
p38MAPKは活性化によって、HSP27をリン酸化することが報告されている(Landry et al.,Biochem.Cell Biol.,703-707,1995)。CD147ヒトキメラ抗体LN22R8によって誘導されるp38のリン酸化が、実際のp38シグナル活性化を促しているか確認するために8)-1の抗体処理サンプルについて、リン酸化をSimple Western assays法(プロテインシンプル ジャパン株式会社、Wes)を用いて評価した。HSP27の検出には、HSP27 抗体(R&D systems社、Cat.AF15801)を用いた。リン酸化HSP27の検出には、Phospho-HSP27 (Ser82)antibody(Cell signaling technology社,2401S)、又はAnti-HSP27(phospho Ser15)抗体(abcam社,Cat.ab76313)を用いた。
p38MAPKの活性化によって、CXCL8のmRNA安定化を介した誘導(Hoffmann et al.,J.Leukoc.Biol.,847-855,2002)、SMAD3/4シグナルの活性化が報告されている(Leovonen et al., PLOS ONE, e57474, 2013)。MIA PaCa-2腫瘍内で、抗体投与後にCXCL8の遺伝子発現が誘導されるか抗体投与後のマウス皮下腫瘍から抽出したRNAを定量的PCR法によって調べた。同様にSMADシグナル活性化によって誘導されることが報告されているRHOB遺伝子(Vasilaki et al.,FASEB Journal,891-905,2010)についてもCD147抗体投与による変動を調べた。内在対照遺伝子として、importin(ipo8)遺伝子とTATA box binding protein(tbp)遺伝子の発現を測定した。抗ヒトCD147ヒトキメラ抗体を投与し、72時間後のMIA PaCa-2腫瘍を採材し、RNA later(QIAGEN社、Cat.76104)で処理した後、RNeasy Mini Kit (250)(QIAGEN社、Cat.74106)を使用し、RNAを抽出した。抗ヒトCD147ヒトキメラ抗体として、実施例1)-14で作製したLN22R8chIgG1、LN22R8chIgG2又はLN22R8chIgG4Pを用いた。定量的RT-PCRには、EXPRESS One-Step SuperScript qRT-PCR kit Universal(Thermofisher scienticic,Cat.11781-01K)を用い、遺伝子定量プローブとして、importin(ipo8) (Thermo Fisher Scientific,Cat.Hs00183533_m1),TATA box binding protein(tbp)(Thermo Fisher Scientific,Cat.Hs00427621_m1),rashomolog family member B(rhoB)(Thermo Fisher Scientific,Cat.hs00269660_s1),interleukin 8(Thermo Fisher Scientific,Cat.Hs00174103_m1)をそれぞれ、用いた。PCR反応に伴う遺伝子特異的な蛍光シグナルの増加をABIPrism7500(Applied Biosystems社)を用いて測定した。
図22(a)にipo8/tbp遺伝子の発現比の平均値(n=3)を標準偏差と併せて示した。
図22(b)にcxcl8/tbp遺伝子の発現比の平均値(n=3)を標準偏差と併せて示した。
図22(c)にrhoB/tbp遺伝子の発現比の平均値(n=3)を標準偏差と併せて示した。
抗ヒトCD147ラット抗体rat_CD147_#110は、実施例2、実施例7に示した通り、ヒトキメラ化によって抗腫瘍効果が増強した。ヒトキメラ抗体LN22R8chIgG1、LN22R8chIgG2又はLN22R8chIgG4P投与後の腫瘍中で観察されたcxcl8、rhoBの誘導について、14)-2と同様にrat_CD147_#110とキメラ抗体#110chIgG4Pを比較した。
SMADシグナルの活性化に重要な分子の一つとして、転写因子、SMAD4が知られている(Zang,et al.,Current Biology,270-276,1997)。一部の膵臓癌では、SMAD4の遺伝子的な欠損によりSMADシグナルが部分的な阻害を受けていることが知られている(Hahn, et al.,Science,350-353,1996)。SMAD4が欠損し、SMADシグナルが部分的に阻害されている膵臓癌細胞株BxPC-3について(Yasutome et al.,Clin.Exp.Metastasis,461-473,2005)、CD147抗体が抗腫瘍効果を示すか調べた。2.5×106細胞のヒト膵臓株BxPC-3(ATCC,Cat.CRL-1687)を100%Matrigel(Corning社、Cat.354234)を含むPBSで懸濁し、6週齢雌のBALB/c-nuマウス(CAnN.Cg-Foxn1nu/CrlCrlj、日本チャールス・リバーより購入)の腋窩部皮下に移植した。腫瘍体積をもとに移植後8日後に群分けを実施し、移植の8、15、22日後にマウス抗ヒトCD147抗体LN22R8R8、又はラット抗ヒトCD147抗体2P10F2 10mg/kgで担癌マウスの腹腔内に投与した(n=5)。対照薬として膵臓癌の治療薬であるゲムシタビン(日本イーライリリー社、ジェムザール(登録商標))を移植の8、15、22日後に200mg/kgで担癌マウスの尾静脈内投与した(n=5)。移植腫瘍の長径及び短径を週2回、電子デジタルノギス(株式会社ミツトヨ製)を用いて測定し、以下に示す計算式により腫瘍体積を算出した。
図24に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。BxPC-3腫瘍は、LN22R8抗体、2P10F2抗体及びゲムシタビンに耐性であった。
複数の膵臓癌細胞株でSMAD4を導入することによってSMADシグナルが回復することが報告されている。SMAD4の導入によって回復したSMADシグナルによりCD147抗体の感受性が増加するか調べた。
Retro-XTM Qベクターキットを利用しBxPC-3のSMAD4安定発現株を作製した。レトロウイルスベクター(タカラバイオ社、Retro-XTM Q Vector Set,Cat.631516)としてキットに含まれるpQCXIPのクローニング部位に人工合成により作製したヒトSMAD4遺伝子を導入し、SMAD4発現レトロウイルスベクターとした。Retro-X Universal Packaging System(タカラバイオ社、Cat.631530)を利用し、BxPC-3にSMAD4発現レトロウイルスベクターを導入し、ピューロマイシン(タカラバイオ社、Cat. 631306)によりウイルス感染により染色体にレトロウイルスが組み込まれ、ピューロマイシン耐性、SMAD4陽性となったBxPC-3細胞を選択し、SMAD4陽性BxPC-3細胞、BxPC-3-SMAD4とした。レトロウイルスベクターpQCXIPを同様に感染させ、ピューロマイシン耐性となったBxPC-3細胞をBxPC-3-mockとした。レトロウイルス感染実験は2回実施し、BxPC-3-mock、BxPC-3-SMAD4について、lot.1,lot.2を作製した。
BxPC-3(ATCC,Cat.CRL-1687)、実施例13)-1で作製したBxPC-3-mock、BxPC-3-SMAD4について、Simple Western assays法(プロテインシンプル ジャパン株式会社、Wes)を用いて解析した。SMAD4陽性対照サンプルとして、MIA PaCa-2を利用した。SMAD4の検出には抗SMAD4抗体(R&D systems,Cat.AF2097)を利用した。GAPDHの検出には、抗GAPDH抗体(Abfrontier,Cat.LF-MA0026)を利用した。CD147の検出には、抗CD147抗体(Abcam,Cat.Ab108317)を利用した。
2.5×106細胞のBxPC-3-mock、又はBxPC-3-SMAD4を100%Matrigel(Corning社、Cat.354234)を含むPBSで懸濁し、5週齢雌のBALB/c-nuマウス(CAnN.Cg-Foxn1nu/CrlCrlj、日本チャールス・リバーより購入)の腋窩部皮下に移植した。腫瘍体積をもとにBxPC-3-mock移植後6日後に、BxPC-3-SMAD4は移植後3日後に群分けをそれぞれ実施し、郡分け実施当日、7、14、21、28日後にヒトキメラ抗ヒトCD147抗体LN22R8R8chIgG2、又はLN22R8R8chIgG4P 10mg/kgで担癌マウスの腹腔内に投与した(n=5)。移植腫瘍の長径及び短径を週2回、電子デジタルノギス(株式会社ミツトヨ製)を用いて測定し、以下に示す計算式により腫瘍体積を算出した。
結果を図25(b)、図25(c)に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。BxPC-3-mock腫瘍は、LN22R8R8chIgG2、又はLN22R8R8chIgG4P抗体に耐性又は、低感受性であった。BxPC-3-SMAD4腫瘍は、LN22R8R8chIgG2、又はLN22R8R8chIgG4P抗体に部分的な感受性を示した。
SMAD4陰性の膵臓癌細胞では、SMAD4を発現させることによって、p38シグナルが増強することが報告されている(Chen et al.,B.M.C.,1471-2407,2014)。BxPC-3-SMAD4腫瘍中のp38MAPKとリン酸化p38MAPKについて、抗ヒトCD147ヒトキメラ抗体投与(抗体投与後72時間後)による変動について、実施例13)-1と同様にSimple Western assays法(プロテインシンプル ジャパン株式会社、Wes)を用いて解析した。実施例1)-18の項と同様に、抗ヒトCD147ヒトキメラ抗体として、LN22R8chIgG4Pを10mg/kg、MIA PaCa-2皮下腫瘍を生着したマウスに投与した。腫瘍組織からのサンプル調製(n=3)には、gentleMACS Octo Dissociator with Heaters(ミルテニー社)を用いた。
ゲムシタビン耐性の膵臓癌腫瘍モデルでのCD147抗体の抗腫瘍効果を調べた。5×106細胞のヒト膵臓株MIA PaCa-2を50%GFR-Matrigel(Corning社、Cat.354230)を含むPBSで懸濁し、5週齢雌のNOD-scidマウス(NOD.CB17-Prkdc<scid>/J、日本チャールス・リバー)の腋窩部皮下に移植した。移植の6日後に膵臓癌の治療薬であるゲムシタビン(日本イーライリリー社)を400mg/kg腹腔内投与し、1週間後に増殖の確認されたゲムシタビン耐性腫瘍を形成したマウスを腫瘍のサイズに基づき群分けを実施し、対照群(n=5)には、ゲムシタビンを移植後13日目(群分け日)、20日目に400mg/kg腹腔内投与した。CD147抗体とゲムシタビン併用投与群(n=5)として、ゲムシタビンを移植後13日目(群分け日)、20日目に400mg/kg腹腔内投与するのに加え、CD147ヒトキメラ抗体LN22R8chIgG4Pを移植後13日目(群分け日)、20日目に10mg/kg腹腔内投与した。移植腫瘍の長径及び短径を週2回、電子デジタルノギス(株式会社ミツトヨ製)を用いて測定し、以下に示す計算式により腫瘍体積を算出した。
15)-1 Hep G2細胞におけるCD147とSMAD4の発現
実施例13)-2と同様に、肝臓癌細胞株HepG2細胞(ATCC, Cat.HB-8065)のCD147とSMAD4発現について確認した。CD147陽性の対照検体として、MIA PaCa-2、BxPC-3(ATCC,Cat.CRL-1687)を用いた。SMAD4陽性の対照検体として、MIA PaCa-2、SMAD4陰性の対照検体として、BxPC-3をそれぞれ用いた。
MIA PaCa-2 0.481
BxPC-3 0.481
Hep G2 2.944
MIA PaCa-2 0.329
BxPC-3 0.003
Hep G2 0.723
HepG2細胞(ATCC,Cat.HB-8065)の細胞表面に発現するCD147をフローサイトメーターにより解析した。ヒトCD147の発現確認には、市販の抗ヒトCD147抗体としてAPCラベルされた抗ヒトCD147マウスIgG1抗体MEM-M6/1-APC(Thermofisher, Cat. MA1-10104)を用いた。マウスIgG1 Isotype control抗体としてmIgG1-APC(ミルテニーバイオ社、Cat. 130-092-214)を用いた。HepG2細胞の懸濁液に、1/10量のMEM-M6/1-APCを加え、4℃、30分間処理した。5%FBSを含むPBS緩衝液で細胞を洗浄後、フローサイトメーター(CantoII、BD Bioscience社)の測定を実施し、図28(a)に結果をまとめた。
肝臓癌細胞HepG2において、抗CD147抗体が、p38MAPKに及ぼす影響を調べるために、抗ヒトCD147ヒトキメラ抗体(LN22R8chIgG4P)又は、実施例6)-4-2で作製した抗ヒトCD147ヒト化抗体(#84H1L2hIgG2、#84H1L2hIg4P、#101H1L2hIgG2、#101H1L2hIgG4P、#110H1L4hIg4P、#131H2L2hIgG2)を10μg/mlで15分間処理したHepG2細胞(ATCC,Cat.HB-8065)について、P38リン酸化をSimple Western assays法(プロテインシンプル ジャパン株式会社、Wes)を用いて評価した。対照サンプルとして、HepG2細胞をヒトIgG(hIgG,Jackson ImmunoResearch社、009-000-003)10μg/mlを同様に処理して解析に供した。p38MAPKの検出には、p38 MAPK rabbit mAb(Cell signaling technology社、Cat.#9212)を用いた。リン酸化p38MAPKの検出には、P-p38 MAPK(T180/Y182)(D3F9)XP rabbit mAb(Cell signaling technology社、#4511S)を用いた。検出されたp38MAPKシグナル対する、リン酸化p38MAPKシグナルの比を図28(b)に示した。
CD147とSMAD4陽性で、抗ヒトCD147抗体によるp38のリン酸化が確認されたヒト肝臓株HepG2(ATCC,Cat.HB-8065)について、抗ヒトCD147ヒトキメラ抗体とヒト化抗体の抗腫瘍効果を検討した。
結果を表7及び図29(a)~(d)に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。
ソラフェニブ投与は、腫瘍増殖の部分的な阻害が観察されたが、腫瘍の消失は認められなかった。LN22R8chIgG4P、#84H1L2hIgG2、#84H1L2hIg4P、#110H1L4hIg4Pは、いずれも腫瘍の消失を伴う優れた抗腫瘍効果を示した。
CD147はT細胞の活性化に伴いCD4陽性、CD8陽性のT細胞で発現が上昇し(Hu et al.,J.Cell.Mol Med.,2132-2143,2010)、一部のCD147抗体では、T細胞の活性化誘導能や増殖を阻害する効果が報告されている(Koch et al.,Int.Immunology,777-786,1999;Chiampanichayakul et al., Immunology 167-178, 2006)。エフェクター機能に依存せず、強い抗腫瘍効果を示す抗ヒトCD147抗体が、T細胞を含む末梢血リンパ球(PBL)に及ぼす影響を調べた。
CD147の発現がT細胞の活性化に伴い上昇するか、ヒトPBMCを用いて調べた。ヒトPBMCを10%FBSを含むRPMI1640培地で、37℃、5%CO2存在下で培養した。培養時に、Dynabeads Human T-Activator CD3/CD28(CD3/CD28 beads,Thermofishers scientific社、Cat.1131D)を添加し増殖を誘導し、4日後にフローサイトメーターの解析を実施し、CD147の発現が変化するか調べた。ヒトCD147の発現確認には、市販の抗ヒトCD147抗体としてAPCラベルされた抗ヒトCD147マウスIgG1抗体MEM-M6/1-APC(CD147-APC,Thermofisher,Cat.MA1-10104)を用いた。マウスIgG1 Isotype control抗体としてmIgG1-APC(ミルテニーバイオ社、Cat.130-092-214)を用いた。ヒトPBMCに含まれるT細胞のCD3,CD4,CD8を検出するために、APC/FireTM 750 anti-human CD3 Antibody(BioLegend社製,Cat.344840)、PerCP/Cy5.5 anti-human CD4(BioLegend社製,Cat.344608)、Brilliant Violet 510 anti-human CD8(BioLegend社製,Cat.344732)を用いた。
ヒト末梢血単核球(PBMC)の増殖における抗ヒトCD147抗体作用を解析した。抗ヒトCD147抗体として、2P10F2chIgG4Pを用いた。CellVue Claret Far Red Fluorescent Cell Linker Kit(sigma、Cat.MIDCLARET-1KT)を用いて、PBMCを蛍光ラベルした後に、10%FBSを含むRPMI1640培地で、37℃、5%CO2存在下で培養した。培養時に、IL-2、Dynabeads Human T-Activator CD3/CD28(CD3/CD28 beads,Thermofishers scientific社、Cat.1131D)を添加し増殖を誘導した際に2P10F2chIgG4P(10μg/ml)を加えて、増殖への影響を調べた。培養3日、5日目に、細胞分裂によって減少したPBMC細胞蛍光シグナルに対しフローサイトメーター(CantoII、BD Bioscience社)を用いた測定を実施し、図31に結果をまとめた。
抗ヒトCD147抗体として、ヒトキメラ抗体#84chIgG1、#84chIgG2、#84chIgG4P、#84chIgG1LALA、#84chIgG4PFALA、#101chIgG4P又は#110chIgG4Pを用いた。ヒト末梢血よりFicoll-Paque PLUS(GEヘルスケア・ジャパン株式会社)を用いて、PBLを調製した。96ウェルプレートに10μg/mlのヒトキメラ抗体をコートした。陰性コントロール抗体としてヒトIgG(hIgG, Jackson ImmunoResearch社、009-000-003)、T細胞の活性化やサイトカイン誘導を誘導する陽性コントロール抗体としてDynabeads Human T-Activator CD3/CD28(CD3/CD28-beads、Thermofishers scientific社、Cat.1131D)を用いた。抗体をコートしたウェルに1x106のPBLを加え、24時間後に、培地中のサイトカイン(IL2,TNFα、INFγ)を測定した。Dynabeads Human T-Activator CD3/CD28は、PBLを加えたウェルに直接添加し、同様に24時間後に、培地中のサイトカイン(IL2,TNFα、INFγ)を測定した。IL2の測定には、Quantikine ELISA Human IL-2(R&D systems,Cat.D2050)を用いた。TNFαの測定には、Amersham TNF-α Human, Biotrak Easy ELISA(GEヘルスケア・ジャパン株式会社,Cat.RPN5967)を用いた。INFγの測定には、Human IFN-γ ELISA development kit(MABTECH社,Cat.3420-1H-6)を用いた。測定は3重に実施し、検出された吸光度の平均値と標準偏差を算出し、図32にまとめた。
17)-1 複合体の結晶化
ヒト化#110H1L4hIgG4PをPepsinにより切断して得られたFab’2を、Dithiothreitolで還元した後、Iodoacetamideによりアルキル化することでFab’断片を取得した。このFab’断片と実施例5)で用いたhCD147v2(22-205)の混合物をSuperdex 10/300GL Increaseカラム(GE Healthcare)を用いたゲルろ過クロマトグラフィーに供与し、複合体画分を取得した。複合体はAmiconUltra15 MWCO 10K(ミリポア社製)で緩衝液(10mM Tris HClpH7.5、50mM NaCl)に置換し、13g/Lに濃縮した。複合体溶液を蒸気拡散法により結晶化した。タンパク質溶液0.5μLに沈殿剤溶液(0.1 M NaMalonate pH 7.0、 12%(w/v)Polyethylene Glycol 3350)を等量加えた溶液を、0.05mLの沈殿剤溶液を入れた密閉容器に両溶液が触れ合わないように収め、25℃で静置した。約1週間後に得られた0.15mm×0.15mm×0.3mmの結晶を30%(w/v)になる様にPolyethylene Glycol 400を加えた沈殿剤溶液に浸してから液体窒素で凍結した。放射光施設フォトンファクトリー(茨城県、日本)のビームラインPF-BL17AにてX線回折データを収集した。得られた回折像からソフトウェアmosflm(CCP4:Collaborative Computational Project No. 4)を用いて回折強度を数値化し、結晶構造因子を求めた。結晶の空間群はP21、結晶の単位格子は(a=64.96Å, b=93.37Å, c=98.31Å、alpha=gamma=90、beta=90.89)であった。
得られた構造因子とFab’断片のホモロジーモデルおよびヒトCD147の既知構造(PDBID:3b5h)の三次元構造座標を用いて分子置換法を行い、位相を決定した。計算にはソフトウェアphaser(CCP4:Collaborative Computational Project No.4)を使用した。結晶は非対称単位に2つの複合体を含んでいた。ソフトウェアRefmac5(CCP4:Collaborative Computational Project No.4)を用いて構造の精密化を行い、ソフトウェアcootを用いてモデルの修正を行った。この操作を繰り返し行い、2.3 Å分解能で最終のR値23%、free R値28%を得た。最終のモデルはヒト化#110H1L4のFab’断片を2つと、それぞれに結合するhCD147v2を含む。なお、片方のhCD147v2についてはアミノ酸残基23-203に相当する電子密度が認められたが、もう片方についてはドメイン1に相当する電子密度は明瞭ではなく、アミノ酸残基103-202に相当する電子密度のみ認められた。
非対称単位に含まれる2つの複合体において共通してヒト化#110H1L4のFab’断片の結合面から4Å以内にあるhCD147v2のアミノ酸残基は以下の通りである:Arg106、Lys108、Ala109、Val110、Lys127、Ser128、Glu129、Ser130、Val131、Pro132、Pro133、Val134、Gln164、Gly165。図41に複合体全体のリボンモデルと表面を、図42にhCD147v2とヒト化#110H1L4との相互作用を示した。
フローサイトメーターによりCD147陽性が確認されたヒト胃癌細胞株KATO III細胞(ATCC,Cat.HTB-103)について、抗ヒトCD147ヒトキメラ抗体とヒト化抗体の抗腫瘍効果を検討した。
結果を図43に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。
未処理群のマウスの腫瘍の平均体積は、移植から24日後に290mm3であるのに対し、LN22R8chIgG4p投与群では、199mm3、#110H1L4hIgG4P投与群のでは、134mm3であり、算出された抗腫瘍効果は、LN22R8chIgG4p投与群で31%、#110H1L4hIgG4P投与群では、54%であった。
フローサイトメーターによりCD147陽性が確認されたヒト慢性骨髄性白血病細胞株KU812細胞(ATCC,Cat.CRL-2099)について、抗ヒトCD147ヒト化抗体の抗腫瘍効果を検討した。
結果を図44に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。
未処理群のマウスの腫瘍の平均体積は、移植から25日後に627mm3であるのに対し、イマチニブ投与群では、328mm3、#110H1L4hIgG4P投与群のでは、6mm3であり、算出された抗腫瘍効果は、イマチニブ投与群で48%、#110H1L4hIgG4P投与群では、97%であった。#110H1L4hIgG4P投与群でのみ、5例中4例で腫瘍の完全縮退が観察された。
フローサイトメーターによりCD147陽性が確認されたヒト大腸癌細胞株SW620細胞(ATCC,Cat.CCL-227)について、ヒト化抗体の抗腫瘍効果を検討した。
結果を図45に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。
未処理群のマウスの腫瘍の平均体積は、移植から21日後に1302mm3であるのに対し、#084H1L2hIg4P投与群では、709mm3、#110H1L4hIgG4P投与群のでは、403mm3であり、算出された抗腫瘍効果は、#084H1L2hIg4P投与群で46%、#110H1L4hIgG4P投与群では、69%であった。
フローサイトメーターによりCD147陽性が確認されたヒト腎臓癌786-Oについて、ヒト化抗体の抗腫瘍効果を検討した。
5×106細胞のヒト腎臓癌786-Oを50%Matrigel(Corning社、Cat.354234)をで懸濁し、5週例雌のNOD-scidマウス(NOD.CB17-Prkdc<scid>/J、日本クレアより購入)の腋窩部皮下に移植した。腫瘍体積をもとに移植後3日後に群分けを実施し、ヒトキメラCD147抗体(LN22R8chIgG4P)、実施例6)-4-2で作製したヒト化CD147抗体(#084H1L2hIg4Pあるいは#110H1L4hIg4P)を10mg/kgで担癌マウスの腹腔内に、群分け後7日ごとに計4回投与した(n=6)。移植腫瘍の長径及び短径を週2回、電子デジタルノギス(株式会社ミツトヨ製)を用いて測定し、以下に示す計算式により腫瘍体積を算出した。
結果を図46に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。
未処理群のマウスの腫瘍の平均体積は、移植から31日後に918mm3であるのに対し、#084H1L2hIg4P投与群では、224mm3、#110H1L4hIgG4P投与群のでは、379mm3であり、算出された抗腫瘍効果は、#084H1L2hIg4P投与群で76%、#110H1L4hIgG4P投与群では、59%であった。
フローサイトメーターによりCD147陽性が確認されたヒトAML細胞株OCI-AML3細胞(DSMZ,Cat.ACC 582)について、ヒト化抗体の抗腫瘍効果を検討した。
結果を図47に示した。グラフには腫瘍体積の変化について、平均値と標準誤差を併せて記載した。
未処理群のマウスの腫瘍の平均体積は、移植から21日後に1533mm3であるのに対し、#110H1L4hIgG4P投与群のでは、394mm3であり、算出された抗腫瘍効果は、#110H1L4hIgG4P投与群では、74%であった。
5×106細胞のヒト膵臓株MIA PaCa-2を50%GFR-Matrigel(Corning社、Cat.354230)を含むPBSで懸濁し、4週例雌のNudeマウス(BALB/c Slc-nu/nu、日本エスエルシー(株)より購入)の腋窩部皮下に移植した。腫瘍体積をもとに群分けをし、移植の7日後に実施例6)-4-2で作製したCD147蛋白質への結合性が異なる3種のヒト化CD147抗体(#110H1L4hIgG4P、#110H13L02hIgG4P及び#110H13L12hIgG4P、表6に結合性を記載)を10 mg/kgで担癌マウスに尾静脈投与した(n=6)。対照薬として膵臓癌の治療薬であるゲムシタビン(日本イーライリリー社より購入)を移植の3、10日後に400 mg/kgで担癌マウスに尾静脈投与した(n=6)。移植腫瘍の長径及び短径を週2回、電子デジタルノギス(株式会社ミツトヨ製)を用いて測定し、以下に示す計算式により腫瘍体積を算出した。
24)-1 既存の抗CD147抗体の抗原結合性評価
競合抗体として、WO2010/036460の4A5、5F6抗体、WO2017/061602のPPAT-082-03の特許記載配列に基づき、精製抗体を調製した。CD147蛋白質との解離定数測定は、Biacore T200(GE Healthcare Bioscience社製)を使用し、Human Antibody Capture Kit(GE Healthcare Bioscience社製)を用いて固定化したAnti-Human IgG(Fc) antibodyに抗体をリガンドとして捕捉(キャプチャー)し、抗原をアナライトとして測定するキャプチャー法にて行った。ランニングバッファーとしてHBS-EP+(GE Healthcare Bioscience社製)、センサーチップとしてCM5(GE Healthcare Bioscience社製)を用いた。チップ上に1μg/mLの競合抗体を10μL/分で60秒間添加した後、実施例2)-5で用いた抗原の希釈系列溶液(0.5~8μg/mL)を流速30μL/分で120秒間添加し、引き続き300秒間の解離相をモニターした。再生溶液として、3M magnesium chloride(GE Healthcare Bioscience社製)を流速20μL/分で30秒間添加した。データの解析には1:1結合モデルを用いて、結合速度定数ka、解離速度定数kd及び解離定数(KD;KD=kd/ka)を算出した。ヒト化CD147抗体(#084H1L2hIgG4Pあるいは#110H1L4hIgG4P)の解離定数は、実施例7)-1の方法で算出した。
実施例6)-4-2で作製したヒト化CD147抗体(#084H1L2hIg4Pあるいは#110H1L4hIg4P)を用いRecombinant Human CD147/Fc(Sino Biological社,Cat.10186-H02H)への結合性を競合ELISAで評価した。競合抗体として、24)-1で調製した4A5、5F6抗体、PPAT-082-03抗体を使用した。競合陰性対照抗体として、ヒトIgG(ジャクソン社、Cat.130093)を用いた。競合陽性対照抗体として、#084H1L2hIg4P抗体に対し、#84H1L2hIgG2抗体、#110H1L4hIg4Pに対し#110chIgG2抗体を用いた。
実施例17)-3で示されたH110H1L4h抗体のエピトープ情報を表8に記載した。実施例1)-9のCD147変異体を用いた解析でエピトープが推定された2P10F2抗体との結合競合性試験結果(実施例2)-8)から、#084H1L2hのエピトープ領域を推定し、表8に記載した。
実施例1)-15の手法に従い、抗CD147抗体のADCC活性を評価した。実施例1)-15の手法と異なる条件として、ADCC標的細胞としてHepG2細胞(ATCC,Cat.HB-8065)を用い、CD147抗体として#110H1L4hIgG4P、#084H1L2hIg4P、4A5、5F6、PPAT-082-03を1μg/mlの濃度で評価した。測定は3重に実施し、平均値、標準偏差を算出した。5%以上の細胞の51Crが検出された場合に、ADCC陽性(+)とした。5%未満の場合をADCC活性陰性(-)とした。結果を表8に示した。
実施例1)-16の手法に従い、抗ヒトCD147抗体による補体依存的な殺細胞活性(CDC活性)を評価した。実施例1)-16の手法と異なる条件として、標的細胞としてヒト肝臓株HepG2細胞(ATCC,Cat.HB-8065)を用い、抗ヒトCD147抗体として、#110H1L4hIgG4P、#084H1L2hIg4P、4A5、5F6およびPPAT-082-03を用い、ウサギ補体を終濃度8%となるように添加し、測定した。測定は3重に実施し、平均値と標準偏差を算出した。30%以上の抗体依存的なCDC活性が認められた抗体については、CDC活性陽性とし、表にCDC(+)と記載した。結果を表8に示した。抗ヒトCD147抗体は、4A5のみCDC活性陽性を示した。#110H1L4hIgG4P、#084H1L2hIg4P、5F6、PPAT-082-03は、CDC陰性であり、ヒトの生体内でCD147を発現している血液細胞などの正常細胞に対して細胞死を誘導する可能性が、CDC活性陽性の4A5よりも低いことが予測される。
24)-5 抗CD147抗体のADCP評価
実施例1)-17の手法に従い、抗CD147抗体のADCP活性を測定した。実施例1)-17の手法と異なる条件として、標的細胞としてヒト肝臓株HepG2細胞(ATCC,Cat.HB-8065)を用い、抗CD147抗体として#110H1L4hIgG4P、#084H1L2hIg4P、4A5、5F6、PPAT-082-03を1μg/mlの濃度で添加し、標識したRAW264.7細胞はADCP標的細胞の等量添加し、ADCP活性を測定した。測定は3重に実施し、平均値、標準偏差を算出し、ヒトIgG処理群より10%未満のADCP活性上昇を弱陽性(±)、10%以上の活性上昇を陽性(+)として表8に示した。
25)-1 抗CD147抗体による血液系細胞凝集
一部の抗CD147抗体は、血球系細胞の凝集を誘導することが報告されている(KasinrerkらImmunology 1999,96(2)p184-192)。血球系細胞の凝集は、重篤な血液毒性を引き起こす可能性があり(Doll,C.,et al.,1994,Curr.Opin.Oncol.,345-350)、治療用抗体としては望ましくない性質である。抗CD147抗体について、細胞集積の活性の違いを調べた。CD147抗体として#110H1L4hIgG4P、#084H1L2hIg4P、4A5、5F6、PPAT-082-03を評価した。陰性の対照抗体としてヒトIgG(hIgG,ChromPure Human IgG,Jackson ImmunoResearch Laboratories社,Cat.009-000-003)を用いた。HEL92.1.7細胞(ATCCより購入、Cat.#TIB-180)を96ウェルUボトムプレート(住友ベークライト社、Cat.MS-9096U)に1ウェルあたり、1600細胞 / 80 uL 10%FBS(ハイクローン社、Cat.SH30084.03)を含むRPMI1640培地(サーモフィッシャーサイエンティフィック社、Cat.11875-093)を添加し、4時間、5%CO2、湿度95%、37度の条件で培養した。各ウェルに20ulの抗CD147抗体溶液(150ug/ml,50ug/ml)を加え、終濃度30、10ug/mlとした。5%CO2、湿度95%、37度の条件で2日間培養し、顕微鏡観察を実施した。
治療用抗体の投与によって、OKT3、TGN1412など一部の抗体では、免疫細胞を活性化させることで、血中のサイトカインが上昇し、重篤なサイトカイン放出症候群を引き起こす(Gaston,R.,Kidney International,1991,141-148;Suntharalingam,G.,et al.,N.Engl.J.Med.2006,1018-1028)。一部のCD147抗体は、免疫細胞に作用し、インターフェロンガンマやインターロイキンー4の産生を増加する作用が報告されている(Hu,J.,et al.,J.Cell.Mol.Med.,2010,2132-2143)。このサイトカイン放出症候群による抗体医薬の毒性は、末梢血を用いたサイトカインリリースアッセイにより予測することができる(Vessillier,S.et al.,J.Immunolol.Methods,2015,43-52)。同様のヒト末梢血サイトカインリリースアッセイによりサイトカイン放出症候群のリスクを評価した。CD147抗体として#110H1L4hIgG4P、#110chIgG4ProFALA、#084H1L2hIg4P、#084H1L2hIg2を用い、比較抗体として、ベバシズマブ(Genentech,Inc.)、トラツズマブ(Roche Pharma AG)、アレムツズマブ(サノフィ株式会社)、抗ヒトCD3抗体(BioLegend Cat.No317326)を用いた。評価した全てのCD147抗体については、ヒト末梢血単核球(6ドナー分をそれぞれ評価)に対し、細胞増殖の亢進は認められず、サイトカインの放出(TNFα、INF-γ、IL-2、IL-6、IL-8、IL-10、MIP-1α)への影響は、サイトカイン放出症候群のリスクが低いベバシズマブよりも弱かった。抗ヒトCD3抗体(OKT3)では、細胞増殖の亢進、サイトカインの放出(TNFα、INF-γ、IL-2、IL-6、IL-8、IL-10、MIP-1α)の亢進が観察された。#110H1L4hIgG4P、#110chIgG4ProFALA、#084H1L2hIg4P、#084H1L2hIg2は、サイトカイン放出症候群の可能性となるサイトカインリリースを誘導しないことが示された。
一部の抗マウスCD147抗体は、マウスに投与した際に、CD147の機能を阻害し、脾臓での赤血球の集積を誘導することで、末梢血中の赤血球量が低下し、貧血を引き起こすことが報告されている(Coste, I. et al., Blood, 2001, 3984-3988)。本発明の中で取得した#110H1L4hIgG4PなどのCD147抗体は、マウスのCD147に結合を示さないため、マウスでの安全性評価は適切ではない。そこで、フローサイトメーターを用いた実験によりヒトおよびサルCD147への結合性が確認された抗CD147抗体として、#110H1L4hIgG4Pをカニクイザルに投与し、安全性を評価した。カニクイザル(雌雄各1例)に#110H1L4hIgG4Pを投与可能最大量である99.2 mg/kgで単回静脈内投与した結果、投与後15日までの観察期間と観察期間終了時の病理組織学的検査では、重篤な毒性(体重および摂餌量の変化、病理組織学的変化)は観察されなかった。#110H1L4hIgG4Pは、カニクイザルに対して毒性を示さず、ヒトの癌治療に利用できる可能性が示された。
癌細胞において、SMAD2/SMAD3/SMAD4依存的な細胞死のシグナルとしてLethal EMTシグナルが知られており、このLethal EMTシグナルは、SMAD4陰性の癌細胞では、通常SMADシグナルによって抑制されている転写因子KLF5蛋白質の発現が増加し、致死性のシグナルを抑制することが報告されている(David,C.Cell,2016,1015-1030)。本発明のCD147抗体は、SMADシグナルを活性化し、SMAD4陽性の細胞に抗腫瘍効果を示すことから、SMADシグナル依存的な細胞死を誘導すると考えた。KLF5がCD147抗体依存的な抗腫瘍効果の感受性に関与するか調べた。
実施例13の方法に準じ、MIA PaCa-2細胞のKLF5安定発現株を作製した。ヒトKLF5のアミノ酸配列及びヌクレオチド配列を、それぞれ、配列番号145及び146に示す。KLF5遺伝子として、(Genscript社、Cat.OHu21278C)に含まれる配列(Ref seq.ID:NM_001730.4)を組み込んだレトロウイルスベクターpQCXIPを作製し、レトロウイルスを作製に用いた。ウイルス感染により染色体にレトロウイルスが組み込まれ、ピューロマイシン耐性、KLF5陽性となったMIA PaCa-2細胞を選択し、KLF5陽性MIA PaCa-2細胞、MIA PaCa-2-KLF5とした。レトロウイルスベクターpQCXIPを同様に感染させ、ピューロマイシン耐性となったMIA PaCa-2細胞をMIA PaCa-2-mockとした。
フローサイトメーターにより、MIA PaCa-2-mockとMIA PaCa-2-KLF5のCD147の発現を確認した。実施例13-2の方法に準じ、MIA PaCa-2-KLF5のKLF5の発現量がMIA PaCa-2-mockから増加していることを確認した。KLF5の検出には、KLF5抗体(CST社、Cat.#51586)を用いた。
26)-3 KLF5を発現したMIA PaCa-2腫瘍のヒト化CD147抗体への感受性
実施例7)-2の方法に準じ、MIA PaCa-2-KLF5とMIA PaCa-2-mockの腫瘍のヒト化CD147抗体#110H1L4hIgG4Pへの感受性を比較した。細胞移植の3日後に実施例6)-4-2で作製したヒト化CD147抗体(#110H1L4hIg4P)を1 mg/kgで担癌マウスに尾静脈投与した(n=6)。7日後に同様に抗体を投与した。対照群の担癌マウスには、PBS緩衝液を同様に尾静脈投与した(n=6)。結果を図50に示す。
配列番号2:ヒトCD147のバリアント1のヌクレオチド配列
配列番号3:ヒトCD147のバリアント2のアミノ酸配列
配列番号4:ヒトCD147のバリアント2のヌクレオチド配列
配列番号5:ヒトSMAD4のアミノ酸配列
配列番号6:ヒトSMAD4のヌクレオチド配列
配列番号7:LN22R8の軽鎖の可変領域のヌクレオチド配列
配列番号8:LN22R8の軽鎖の可変領域のアミノ酸配列
配列番号9:LN22R8の重鎖の可変領域のヌクレオチド配列
配列番号10:LN22R8の重鎖の可変領域のアミノ酸配列
配列番号11:LN22R8のCDRL1のアミノ酸配列
配列番号12:LN22R8のCDRL2のアミノ酸配列
配列番号13:LN22R8のCDRL3のアミノ酸配列
配列番号14:LN22R8のCDRH1のアミノ酸配列
配列番号15:LN22R8のCDRH2のアミノ酸配列
配列番号16:LN22R8のCDRH3のアミノ酸配列
配列番号17:2P10F2の軽鎖の可変領域のヌクレオチド配列
配列番号18:2P10F2の軽鎖の可変領域のアミノ酸配列
配列番号19:2P10F2の重鎖の可変領域のヌクレオチド配列
配列番号20:2P10F2の重鎖の可変領域のアミノ酸配列
配列番号21:2P10F2のCDRL1のアミノ酸配列
配列番号22:2P10F2のCDRL2のアミノ酸配列
配列番号23:2P10F2のCDRL3のアミノ酸配列
配列番号24:2P10F2のCDRH1のアミノ酸配列
配列番号25:2P10F2のCDRH2のアミノ酸配列
配列番号26:2P10F2のCDRH3のアミノ酸配列
配列番号27:ヒト軽鎖シグナル配列及びヒトκ鎖定常領域をコードするDNA配列を含むDNA断片
配列番号28:ヒト重鎖シグナル配列及びヒトIgG1定常領域のアミノ酸をコードするDNA配列を含むDNA断片
配列番号29:ヒト重鎖シグナル配列及びヒトIgG2定常領域のアミノ酸をコードするDNA配列を含むDNA断片
配列番号30:ヒトキメラLN22R8の軽鎖のヌクレオチド配列
配列番号31:ヒトキメラLN22R8の軽鎖のアミノ酸配列
配列番号32:ヒトキメラLN22R8の重鎖IgG1タイプのヌクレオチド配列
配列番号33:ヒトキメラLN22R8の重鎖IgG1タイプのアミノ酸配列
配列番号34:ヒトキメラLN22R8の重鎖IgG2タイプのヌクレオチド配列
配列番号35:ヒトキメラLN22R8の重鎖IgG2タイプのアミノ酸配列
配列番号36:ヒトキメラLN22R8の重鎖IgG4Pタイプのアミノ酸配列をコードするDNA配列を含むDNA断片
配列番号37:ヒトキメラLN22R8の重鎖IgG4Pタイプのアミノ酸配列
配列番号38:ヒト重鎖シグナル配列及びヒトIgG1LALA定常領域のアミノ酸をコードするDNA配列を含むDNA断片
配列番号39:ヒト重鎖シグナル配列及びヒトIgG4P定常領域のアミノ酸をコードするDNA配列を含むDNA断片
配列番号40:ヒトキメラ2P10F2の軽鎖のヌクレオチド配列
配列番号41:ヒトキメラ2P10F2の軽鎖のアミノ酸配列
配列番号42:ヒトキメラ2P10F2の重鎖IgG1LALAタイプのヌクレオチド配列
配列番号43:ヒトキメラ2P10F2の重鎖IgG1LALAタイプのアミノ酸配列
配列番号44:ヒトキメラ2P10F2の重鎖IgG2タイプのヌクレオチド配列
配列番号45:ヒトキメラ2P10F2の重鎖IgG2タイプのアミノ酸配列
配列番号46:ヒトキメラ2P10F2の重鎖IgG4Pタイプのヌクレオチド配列
配列番号47:ヒトキメラ2P10F2の重鎖IgG4Pタイプのアミノ酸配列
配列番号48:rat_CD147_#84(r#84)の軽鎖の可変領域のヌクレオチド配列
配列番号49:rat_CD147_#84(r#84)の軽鎖の可変領域のアミノ酸配列
配列番号50:rat_CD147_#84(r#84)の重鎖の可変領域のヌクレオチド配列
配列番号51:rat_CD147_#84(r#84)の重鎖の可変領域のアミノ酸配列
配列番号52:rat_CD147_#84(r#84)のCDRL1のアミノ酸配列
配列番号53:rat_CD147_#84(r#84)のCDRL2のアミノ酸配列
配列番号54:rat_CD147_#84(r#84)のCDRL3のアミノ酸配列
配列番号55:rat_CD147_#84(r#84)のCDRH1のアミノ酸配列
配列番号56:rat_CD147_#84(r#84)のCDRH2のアミノ酸配列
配列番号57:rat_CD147_#84(r#84)のCDRH3のアミノ酸配列
配列番号58:rat_CD147_#101(r#101)の軽鎖の可変領域のヌクレオチド配列
配列番号59:rat_CD147_#101(r#101)の軽鎖の可変領域のアミノ酸配列
配列番号60:rat_CD147_#101(r#101)の重鎖の可変領域のヌクレオチド配列
配列番号61:rat_CD147_#101(r#101)の重鎖の可変領域のアミノ酸配列
配列番号62:rat_CD147_#101(r#101)のCDRL1のアミノ酸配列
配列番号63:rat_CD147_#101(r#101)のCDRL2のアミノ酸配列
配列番号64:rat_CD147_#101(r#101)のCDRL3のアミノ酸配列
配列番号65:rat_CD147_#101(r#101)のCDRH1のアミノ酸配列
配列番号66:rat_CD147_#101(r#101)のCDRH2のアミノ酸配列
配列番号67:rat_CD147_#101(r#101)のCDRH3のアミノ酸配列
配列番号68:rat_CD147_#110(r#110)の軽鎖の可変領域のヌクレオチド配列
配列番号69:rat_CD147_#110(r#110)の軽鎖の可変領域のアミノ酸配列
配列番号70:rat_CD147_#110(r#110)の重鎖の可変領域のヌクレオチド配列
配列番号71:rat_CD147_#110(r#110)の重鎖の可変領域のアミノ酸配列
配列番号72:rat_CD147_#110(r#110)のCDRL1のアミノ酸配列
配列番号73:rat_CD147_#110(r#110)のCDRL2のアミノ酸配列
配列番号74:rat_CD147_#110(r#110)のCDRL3のアミノ酸配列
配列番号75:rat_CD147_#110(r#110)のCDRH1のアミノ酸配列
配列番号76:rat_CD147_#110(r#110)のCDRH2のアミノ酸配列
配列番号77:rat_CD147_#110(r#110)のCDRH3のアミノ酸配列
配列番号78:rat_CD147_#131(r#131)の軽鎖の可変領域のヌクレオチド配列
配列番号79:rat_CD147_#131(r#131)の軽鎖の可変領域のアミノ酸配列
配列番号80:rat_CD147_#131(r#131)の重鎖の可変領域のヌクレオチド配列
配列番号81:rat_CD147_#131(r#131)の重鎖の可変領域のアミノ酸配列
配列番号82:rat_CD147_#131(r#131)のCDRL1のアミノ酸配列
配列番号83:rat_CD147_#131(r#131)のCDRL2のアミノ酸配列
配列番号84:rat_CD147_#131(r#131)のCDRL3のアミノ酸配列
配列番号85:rat_CD147_#131(r#131)のCDRH1のアミノ酸配列
配列番号86:rat_CD147_#131(r#131)のCDRH2のアミノ酸配列
配列番号87:rat_CD147_#131(r#131)のCDRH3のアミノ酸配列
配列番号88:ヒト重鎖シグナル配列及びヒトIgG4PFALA定常領域のアミノ酸をコードするDNA配列を含むDNA断片
配列番号89:ヒトキメラrat_CD147_#84の軽鎖のヌクレオチド配列
配列番号90:ヒトキメラrat_CD147_#84の軽鎖のアミノ酸配列
配列番号91:ヒトキメラrat_CD147_#84の重鎖IgG1タイプのヌクレオチド配列
配列番号92:ヒトキメラrat_CD147_#84の重鎖IgG1タイプのアミノ酸配列
配列番号93:ヒトキメラrat_CD147_#84の重鎖IgG2タイプのヌクレオチド配列
配列番号94:ヒトキメラrat_CD147_#84の重鎖IgG2タイプのアミノ酸配列
配列番号95:ヒトキメラrat_CD147_#84の重鎖IgG4Pタイプのヌクレオチド配列
配列番号96:ヒトキメラrat_CD147_#84の重鎖IgG4Pタイプのアミノ酸配列
配列番号97:ヒトキメラrat_CD147_#84の重鎖IgG1LALAタイプのヌクレオチド配列
配列番号98:ヒトキメラrat_CD147_#84の重鎖IgG1LALAタイプのアミノ酸配列
配列番号99:ヒトキメラrat_CD147_#84の重鎖IgG4PFALAタイプのヌクレオチド配列
配列番号100:ヒトキメラrat_CD147_#84の重鎖IgG4PFALAタイプのアミノ酸配列
配列番号101:ヒトキメラrat_CD147_#101の軽鎖のヌクレオチド配列
配列番号102:ヒトキメラrat_CD147_#101の軽鎖のアミノ酸配列
配列番号103:ヒトキメラrat_CD147_#101の重鎖IgG2のヌクレオチド配列
配列番号104:ヒトキメラrat_CD147_#101の重鎖IgG2のアミノ酸配列
配列番号105:ヒトキメラrat_CD147_#101の重鎖IgG4Pのヌクレオチド配列
配列番号106:ヒトキメラrat_CD147_#101の重鎖IgG4Pのアミノ酸配列
配列番号107:ヒトキメラrat_CD147_#101の重鎖IgG4PFALAのヌクレオチド配列
配列番号108:ヒトキメラrat_CD147_#101の重鎖IgG4PFALAのアミノ酸配列
配列番号109:ヒトキメラrat_CD147_#110の軽鎖のヌクレオチド配列
配列番号110:ヒトキメラrat_CD147_#110の軽鎖のアミノ酸配列
配列番号111:ヒトキメラrat_CD147_#110の重鎖IgG2のヌクレオチド配列
配列番号112:ヒトキメラrat_CD147_#110の重鎖IgG2のアミノ酸配列
配列番号113:ヒトキメラrat_CD147_#110の重鎖IgG4Pのヌクレオチド配列
配列番号114:ヒトキメラrat_CD147_#110の重鎖IgG4Pのアミノ酸配列
配列番号115:ヒトキメラrat_CD147_#110の重鎖IgG4PFALAのヌクレオチド配列
配列番号116:ヒトキメラrat_CD147_#110の重鎖IgG4PFALAのアミノ酸配列
配列番号117:ヒトキメラrat_CD147_#131の軽鎖のヌクレオチド配列
配列番号118:ヒトキメラrat_CD147_#131の軽鎖のアミノ酸配列
配列番号119:ヒトキメラrat_CD147_#131の重鎖IgG2のヌクレオチド配列
配列番号120:ヒトキメラrat_CD147_#131の重鎖IgG2のアミノ酸配列
配列番号121:ヒトキメラrat_CD147_#131の重鎖IgG4Pのヌクレオチド配列
配列番号122:ヒトキメラrat_CD147_#131の重鎖IgG4Pのアミノ酸配列
配列番号123:#84H1hIgG2のアミノ酸配列
配列番号124:#84H1hIgG2のヌクレオチド配列
配列番号125:#84H1hIgG4Pのアミノ酸配列
配列番号126:#84H1hIgG4Pのヌクレオチド配列
配列番号127:#84L2hのアミノ酸配列
配列番号128:#84L2hのヌクレオチド配列
配列番号129:#101H1hIgG2のアミノ酸配列
配列番号130:#101H1hIgG2のヌクレオチド配列
配列番号131:#101H1hIgG4Pのアミノ酸配列
配列番号132:#101H1hIgG4Pのヌクレオチド配列
配列番号133:#101L2hのアミノ酸配列
配列番号134:#101L2hのヌクレオチド配列
配列番号135:#110H1hIgG4Pのアミノ酸配列
配列番号136:#110H1hIgG4Pのヌクレオチド配列
配列番号137:#110L4hのアミノ酸配列
配列番号138:#110L4hのヌクレオチド配列
配列番号139:#131H2hIgG2のアミノ酸配列
配列番号140:#131H2hIgG2のヌクレオチド配列
配列番号141:#131L2hのアミノ酸配列
配列番号142:#131L2hのヌクレオチド配列
配列番号143:ヒトCD147v1のmu3領域
配列番号144:カニクイサルCD147のmu3領域
配列番号145:ヒトKLF5のアミノ酸配列
配列番号146:ヒトKLF5のヌクレオチド配列
配列番号147:#110H13hIgG4Pのアミノ酸配列
配列番号148:#110H13hIgG4Pのヌクレオチド配列
配列番号149:#110L2hのアミノ酸配列
配列番号150:#110L2hのヌクレオチド配列
配列番号151:#110L12hのアミノ酸配列
配列番号152:#110L12hのヌクレオチド配列
Claims (71)
- 以下の(A)~(F)からなる群から選択される少なくともいずれか1つの抗体と、ヒトCD147への結合に対し競合し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(A)配列番号71に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号69に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(B)配列番号51に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号49に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(C)配列番号61に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号59に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(D)配列番号81に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号79に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(E)配列番号10に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号8に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、及び
(F)配列番号20に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号18に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体。 - 以下の(A)~(F)からなる群から選択される少なくともいずれか1つの抗体が結合するエピトープに結合し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(A)配列番号71に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号69に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(B)配列番号51に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号49に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(C)配列番号61に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号59に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(D)配列番号81に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号79に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、
(E)配列番号10に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号8に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体、及び
(F)配列番号20に示されるアミノ酸配列からなる重鎖可変領域、及び配列番号18に示されるアミノ酸配列からなる軽鎖可変領域を含む抗体。 - ADCC活性が低下又は欠失した、請求項1又は2に記載のヒトCD147抗体又は該抗体の抗原結合断片。
- CDC活性が低下又は欠失した、請求項1~3のいずれか1項に記載のヒトCD147抗体又は該抗体の抗原結合断片。
- ADCP活性が低下又は欠失した、請求項1~4のいずれか1項に記載のヒトCD147抗体又は該抗体の抗原結合断片。
- 配列番号3の106番目のアルギニン(Arg)から165番目のグリシン(Gly)の残基を含むエピトープに結合する、請求項1~5のいずれか1項に記載の抗体又は該抗体の抗原結合断片。
- 配列番号3に記載のアミノ酸配列中の、106番目のアルギニン(Arg)、108番目のリシン(Lys)、109番目のアラニン(Ala)、110番目のバリン(Val)、127番目のリシン(Lys)、128番目のセリン(Ser)、129番目のグルタミン酸(Glu)、130番目のセリン(Ser)、131番目のバリン(Val)、132番目のプロリン(Pro)、133番目のプロリン(Pro)、134番目のバリン(Val)、164番目のグルタミン(Gln)及び165番目のグリシン(Gly)の各残基を含むエピトープに結合する、請求項1~6のいずれか1項に記載の抗体又は該抗体の抗原結合断片。
- 重鎖配列が、CDRH1、CDRH2及びCDRH3を有する可変領域を含み、前記CDRH1は配列番号75に示されるアミノ酸配列からなり、前記CDRH2は配列番号76に示されるアミノ酸配列からなり、前記CDRH3は、配列番号77に示されるアミノ酸配列からなること;及び
軽鎖配列がCDRL1、CDRL2及びCDRL3を有する可変領域を含み、前記CDRL1は配列番号72に示されるアミノ酸配列からなり、前記CDRL2は配列番号73に示されるアミノ酸配列からなり、前記CDRL3は、配列番号74に示されるアミノ酸配列からなること;
を特徴とする、請求項1~7のいずれか1項に記載の抗体又は該抗体の抗原結合断片。 - 配列番号143に記載のアミノ酸配列、又は、配列番号143の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列を含むエピトープに結合する、請求項1~5のいずれか1項に記載の抗体又は該抗体の抗原結合断片。
- 重鎖配列が、CDRH1、CDRH2及びCDRH3を有する可変領域を含み、前記CDRH1は配列番号55に示されるアミノ酸配列からなり、前記CDRH2は配列番号56に示されるアミノ酸配列からなり、前記CDRH3は、配列番号57に示されるアミノ酸配列からなること;及び
軽鎖配列がCDRL1、CDRL2及びCDRL3を有する可変領域を含み、前記CDRL1は配列番号52に示されるアミノ酸配列からなり、前記CDRL2は配列番号53に示されるアミノ酸配列からなり、前記CDRL3は、配列番号54に示されるアミノ酸配列からなること;
を特徴とする、請求項1~5又は9のいずれか1項に記載の抗体又は該抗体の抗原結合性断片。 - 重鎖配列が、CDRH1、CDRH2及びCDRH3を有する可変領域を含み、前記CDRH1は配列番号65に示されるアミノ酸配列からなり、前記CDRH2は配列番号66に示されるアミノ酸配列からなり、前記CDRH3は、配列番号67に示されるアミノ酸配列からなること;及び
軽鎖配列がCDRL1、CDRL2及びCDRL3を有する可変領域を含み、前記CDRL1は配列番号62に示されるアミノ酸配列からなり、前記CDRL2は配列番号63に示されるアミノ酸配列からなり、前記CDRL3は、配列番号64に示されるアミノ酸配列からなること;
を特徴とする、請求項1~5又は9のいずれか1項に記載の抗体又は該抗体の抗原結合性断片。 - 重鎖配列が、CDRH1、CDRH2及びCDRH3を有する可変領域を含み、前記CDRH1は配列番号85に示されるアミノ酸配列からなり、前記CDRH2は配列番号86に示されるアミノ酸配列からなり、前記CDRH3は、配列番号87に示されるアミノ酸配列からなること;及び
軽鎖配列がCDRL1、CDRL2及びCDRL3を有する可変領域を含み、前記CDRL1は配列番号82に示されるアミノ酸配列からなり、前記CDRL2は配列番号83に示されるアミノ酸配列からなり、前記CDRL3は、配列番号84に示されるアミノ酸配列からなること;
を特徴とする、請求項1~5又は9のいずれか1項に記載の抗体又は該抗体の抗原結合性断片。 - Fab、F(ab’)2、Fab’及びFvからなる群から選択される、請求項1~12のいずれか1項に記載の抗体の抗原結合断片。
- scFvであることを特徴とする、請求項1~12のいずれか1項に記載の抗体。
- キメラ抗体であることを特徴とする、請求項1~12のいずれか1項に記載の抗体文は該抗体の抗原結合断片。
- ヒ卜化されていることを特徴とする、請求項1~12のいずれか1項に記載の抗体文は該抗体の抗原結合断片。
- 重鎖がヒ卜免疫グ口ブリンG1重鎖、ヒ卜免疫グ口ブリンG2重鎖又はヒ卜免疫グ口ブリンG4重鎖の定常領域を含み、軽鎖がヒ卜免疫グ口プリンκ軽鎖の定常領域を含む、請求項1~16のいずれか1項に記載の抗体。
- 重鎖がヒ卜免疫グ口ブリンG4重鎖の定常領域を含む、請求項17に記載の抗体。
- ヒ卜免疫グ口ブリンG4重鎖の定常領域において、EUインデックスにより示される228番目のセリン(Ser)がプ口リン(Pro)により置換された、請求項18に記載の抗体。
- ヒ卜免疫グ口ブリンG4重鎖の定常領域において、EUインデックスにより示される234番目のフェニルアラニン(Phe)がアラニン(Ala)へ置換され、235番目のロイシン(Leu)がアラニン(Ala)に置換されている、請求項18の抗体。
- ヒ卜免疫グ口ブリンG4重鎖の定常領域において、EUインデックスにより示される228番目のセリン(Ser)がプ口リン(Pro)により置換され、234番目のフェニルアラニン(Phe)がアラニン(Ala)へ置換され、235番目のロイシン(Leu)がアラニン(Ala)に置換されている、請求項18の抗体。
- 重鎖がヒ卜免疫グ口プリンG2重鎖の定常領域を含む、請求項17に記載の抗体。
- 以下の(c)及び(d)を有し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(c)以下の(c1)~(c4)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(c1)配列番号135に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域;
(c2)配列番号147に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域;
(c3)(c1)又は(c2)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(c4)(c1)~(c3)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(d)以下の(d1)~(d5)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(d1)配列番号137に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(d2)配列番号149に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(d3)配列番号151に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(d4)(d1)~(d3)のいずれか1に記載の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(d5)(d1)~(d4)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。 - 配列番号135に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号137に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、請求項23に記載の抗体又は該抗体の抗原結合性断片。
- 配列番号135に示されるアミノ酸配列の20~463番目のアミノ酸残基からなる重鎖、及び、配列番号137に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、請求項23に記載の抗体又は該抗体の抗原結合性断片。
- 配列番号147に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号149に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、請求項23に記載の抗体又は該抗体の抗原結合性断片。
- 配列番号147に示されるアミノ酸配列の20~463番目のアミノ酸残基からなる重鎖、及び、配列番号149に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、請求項23に記載の抗体又は該抗体の抗原結合性断片。
- 配列番号147に示されるアミノ酸配列の20~136番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号151に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、請求項23に記載の抗体又は該抗体の抗原結合性断片。
- 配列番号147に示されるアミノ酸配列の20~463番目のアミノ酸残基からなる重鎖、及び、配列番号151に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、請求項23に記載の抗体又は該抗体の抗原結合性断片。
- 以下の(a)及び(b)を有し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(a)以下の(a1)~(a4)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(a1)配列番号123に示されるアミノ酸配列の20~140番目のアミノ酸残基からなる重鎖可変領域;
(a2)配列番号125に示されるアミノ酸配列の20~140番目のアミノ酸残基からなる重鎖可変領域;
(a3)(a1)又は(a2)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(a4)(a1)~(a3)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(b)以下の(b1)~(b3)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(b1)配列番号127に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(b2)(b1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(b3)(b1)又は(b2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。 - 配列番号123に示されるアミノ酸配列の20~140番目のアミノ酸残基からなる重鎖可変領域又は配列番号125に示されるアミノ酸配列の20~140番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号127に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、請求項30に記載の抗体又は該抗体の抗原結合性断片。
- 配列番号123に示されるアミノ酸配列の20~466番目のアミノ酸残基からなる重鎖又は配列番号125に示されるアミノ酸配列の20~467番目のアミノ酸残基からなる重鎖、及び、配列番号127に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、請求項30に記載の抗体又は該抗体の抗原結合性断片。
- 以下の(e)及び(f)を有し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(e)以下の(e1)~(e4)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(e1)配列番号129に示されるアミノ酸配列の20~137番目のアミノ酸残基からなる重鎖可変領域;
(e2)配列番号131に示されるアミノ酸配列の20~137番目のアミノ酸残基からなる重鎖可変領域;
(e3)(e1)又は(e2)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(e4)(e1)~(e3)のいずれか1に記載の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(f)以下の(f1)~(f3)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(f1)配列番号133に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(f2)(f1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(f3)(f1)又は(f2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。 - 配列番号129に示されるアミノ酸配列の20~137番目のアミノ酸残基からなる重鎖可変領域又は配列番号131に示されるアミノ酸配列の20~137番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号133に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、請求項33に記載の抗体又は該抗体の抗原結合性断片。
- 配列番号129に示されるアミノ酸配列の20~463番目のアミノ酸残基からなる重鎖又は配列番号131に示されるアミノ酸配列の20~464番目のアミノ酸残基からなる重鎖、及び、配列番号133に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、請求項33に記載の抗体又は該抗体の抗原結合性断片。
- 以下の(g)及び(h)を有し、かつ、CD147を介したシグナル伝達を活性化することを特徴とする、ヒトCD147抗体又は該抗体の抗原結合断片:
(g)以下の(g1)~(g3)からなる群から選択されるいずれか1に記載の重鎖可変領域:
(g1)配列番号139に示されるアミノ酸配列の20~138番目のアミノ酸残基からなる重鎖可変領域;
(g2)(g1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(g3)(g1)又は(g2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、並びに、
(h)以下の(h1)~(h3)からなる群から選択されるいずれか1に記載の軽鎖可変領域:
(h1)配列番号141に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域;
(h2)(h1)の配列において各CDR配列以外のフレームワーク領域の配列に対して少なくとも95%以上の配列同一性を有するアミノ酸配列;及び
(h3)(h1)又は(h2)の配列における各CDR配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。 - 配列番号139に示されるアミノ酸配列の20~138番目のアミノ酸残基からなる重鎖可変領域、及び、配列番号141に示されるアミノ酸配列の21~128番目のアミノ酸残基からなる軽鎖可変領域を含む、請求項36に記載の抗体又は該抗体の抗原結合性断片。
- 配列番号139に示されるアミノ酸配列の20~464番目のアミノ酸残基からなる重鎖、及び、配列番号141に示されるアミノ酸配列の21~234番目のアミノ酸残基からなる軽鎖を含む、請求項36に記載の抗体又は該抗体の抗原結合性断片。
- ADCC活性が低下又は欠失した、請求項23~38のいずれか1項に記載のヒトCD147抗体又は該抗体の抗原結合断片。
- CDC活性が低下又は欠失した、請求項23~39のいずれか1項に記載のヒトCD147抗体又は該抗体の抗原結合断片。
- ADCP活性が低下又は欠失した、請求項23~40のいずれか1項に記載のヒトCD147抗体又は該抗体の抗原結合断片。
- 請求項1~41のいずれか1項に記載の抗体又は該抗体の抗原結合断片の少なくともいずれか一つを含有することを特徴とする、医薬組成物。
- 抗腫瘍剤である、請求項42に記載の医薬組成物。
- 腫瘍が、CD147を発現する腫瘍である、請求項43に記載の医薬組成物。
- 腫瘍が、膵臓癌、肝癌、胃癌、大腸癌、腎癌、乳癌、子宮癌、卵巣癌、肺癌、リンパ腫、甲状腺癌、皮膚癌、頭頸部癌、肉腫、前立腺癌、膀胱癌、脳腫瘍、消化管間質腫瘍(GIST)、白血病、急性骨髄性白血病(AML)、慢性骨髄性白血病(CML)、慢性リンパ性白血病(CLL)、急性リンパ性白血病(ALL)、悪性リンパ腫、B細胞リンパ腫、非ホジキンリンパ腫又はびまん性大細胞型B細胞リンパ腫(DLBCL)である、請求項43又は44に記載の医薬組成物。
- 腫瘍が、膵臓癌、肝癌、胃癌、大腸癌、腎癌、白血病、急性骨髄性白血病(AML)、慢性骨髄性白血病(CML)、慢性リンパ性白血病(CLL)、急性リンパ性白血病(ALL)、悪性リンパ腫、B細胞リンパ腫、非ホジキンリンパ腫又はびまん性大細胞型B細胞リンパ腫(DLBCL)である、請求項43~45のいずれか1項に記載の医薬組成物。
- 腫瘍がSMAD4陽性の腫瘍又はKLF5の発現が低下又は欠失した腫瘍である、請求項43~46のいずれか1項に記載の医薬組成物。
- さらに別の抗腫瘍剤を含有する、請求項42~47のいずれか1項に記載の医薬組成物。
- 請求項1~41のいずれか1項に記載の抗体若しくは該抗体の抗原結合性断片又は請求項42~48のいずれか1項に記載の医薬組成物を患者に投与することを特徴とする腫瘍の治療方法。
- 腫瘍が、CD147を発現する腫瘍である、請求項49に記載の治療方法。
- 腫瘍が、膵臓癌、肝癌、胃癌、大腸癌、腎癌、乳癌、子宮癌、卵巣癌、肺癌、リンパ腫、甲状腺癌、皮膚癌、頭頸部癌、肉腫、前立腺癌、膀胱癌、脳腫瘍、消化管間質腫瘍(GIST)、白血病、急性骨髄性白血病(AML)、慢性骨髄性白血病(CML)、慢性リンパ性白血病(CLL)、急性リンパ性白血病(ALL)、悪性リンパ腫、B細胞リンパ腫、非ホジキンリンパ腫又はびまん性大細胞型B細胞リンパ腫(DLBCL)である、請求項49又は50に記載の治療方法。
- 腫瘍が、膵臓癌、肝癌、胃癌、大腸癌、腎癌、白血病、急性骨髄性白血病(AML)、慢性骨髄性白血病(CML)、慢性リンパ性白血病(CLL)、急性リンパ性白血病(ALL)、悪性リンパ腫、B細胞リンパ腫、非ホジキンリンパ腫又はびまん性大細胞型B細胞リンパ腫(DLBCL)である、請求項49~51のいずれか1項に記載の治療方法。
- 腫瘍がSMAD4陽性の腫瘍又はKLF5の発現が低下又は欠失した腫瘍である、請求項49~52のいずれか1項に記載の治療方法。
- 別の抗腫瘍剤と組み合わせて投与される、請求項49~53のいずれか1項に記載の治療方法。
- 請求項1~41のいずれか1項に記載の抗体又は当該抗体の機能性断片をコードするポリヌクレオチド。
- 以下の(j1)~(j3)からなる群から選択されるいずれか1に記載のポリヌクレオチドを含む請求項55に記載のポリヌクレオチド:
(j1)配列番号75に記載のアミノ酸配列からなるCDRH1、配列番号76に記載のアミノ酸配列からなるCDRH2及び配列番号77に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに、配列番号72に記載のアミノ酸配列からなるCDRL1、配列番号73に記載のアミノ酸配列からなるCDRL2及び配列番号74に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、
(j2)(j1)に記載のヌクレオチド配列と少なくとも95%の配列同一性を有するポリヌクレオチド;及び
(j3)(j1)又は(j2)に記載のポリヌクレオチドにおいて1~数個のヌクレオチドが置換、欠失又は付加されたポリヌクレオチド。 - 以下の(i1)~(i3)からなる群から選択されるいずれか1に記載のポリヌクレオチドを含む請求項55に記載のポリヌクレオチド:
(i1)配列番号55に記載のアミノ酸配列からなるCDRH1、配列番号56に記載のアミノ酸配列からなるCDRH2及び配列番号57に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに、配列番号52に記載のアミノ酸配列からなるCDRL1、配列番号53に記載のアミノ酸配列からなるCDRL2及び配列番号54に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド;
(i2)(i1)に記載のヌクレオチド配列と少なくとも95%の配列同一性を有するポリヌクレオチド;及び
(i3)(i1)又は(i2)に記載のポリヌクレオチドにおいて1~数個のヌクレオチドが置換、欠失又は付加されたポリヌクレオチド。 - 以下の(k1)~(k3)からなる群から選択されるいずれか1に記載のポリヌクレオチドを含む請求項55に記載のポリヌクレオチド:
(k1)配列番号65に記載のアミノ酸配列からなるCDRH1、配列番号66に記載のアミノ酸配列からなるCDRH2及び配列番号67に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに、配列番号62に記載のアミノ酸配列からなるCDRL1、配列番号63に記載のアミノ酸配列からなるCDRL2及び配列番号64に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、
(k2)(k1)に記載のヌクレオチド配列と少なくとも95%の配列同一性を有するポリヌクレオチド;及び
(k3)(k1)又は(k2)に記載のポリヌクレオチドにおいて1~数個のヌクレオチドが置換、欠失又は付加されたポリヌクレオチド。 - 以下の(m1)~(m3)からなる群から選択されるいずれか1に記載のポリヌクレオチドを含む請求項53に記載のポリヌクレオチド:
(m1)配列番号85に記載のアミノ酸配列からなるCDRH1、配列番号86に記載のアミノ酸配列からなるCDRH2及び配列番号87に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに、配列番号82に記載のアミノ酸配列からなるCDRL1、配列番号83に記載のアミノ酸配列からなるCDRL2及び配列番号84に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、
(m2)(m1)に記載のヌクレオチド配列と少なくとも95%の配列同一性を有するポリヌクレオチド;及び
(m3)(m1)又は(m2)に記載のポリヌクレオチドにおいて1~数個のヌクレオチドが置換、欠失又は付加されたポリヌクレオチド。 - 請求項55~59のいずれか1項に記載のポリヌクレオチドを含有する発現ベクター。
- 請求項60に記載の発現ベクターにより形質転換された宿主細胞。
- 請求項61に記載の宿主細胞を培養し、培養産物から目的の抗体又は当該抗体の機能性断片を採取する工程を含む、請求項1~41のいずれか1項に記載の抗体又は当該抗体の機能性断片の製造方法。
- CD147を介したシグナル伝達の活性化が、p38MAPKの活性化及び/又はSMAD4の活性化である、請求項1~41のいずれか1項に記載の抗体又は当該抗体の機能性断片。
- p38MAPKの活性化及び/又はSMAD4の活性化が、p38MAPKの発現量の増加、p38MAPKのリン酸化、HSP27のリン酸化、CXCL8発現量の増加、rhoB発現量の増加、KLF5 mRNAの低下又はKLF5蛋白質発現量の低下である、請求項63に記載の抗体又は当該抗体の機能性断片。
- 請求項63又は64に記載の抗体又は該抗体の抗原結合性断片を投与することを特徴とする腫瘍の治療方法。
- 癌患者由来の生物学的試料を用い、該生物学的試料中に含まれるSMAD4の発現又はKLF5の発現を検出し、SMAD4が検出された患者又はKLF5の発現低下若しくは欠失が検出された患者を、請求項1~41のいずれか1項に記載の抗体若しくは当該抗体の機能性断片又は請求項42~48のいずれか1項に記載の医薬組成物による癌の治療への応答性があると判定することを含む、癌の治療への応答性を予測する方法。
- 癌患者由来の生物学的試料を用い、該生物学的試料中におけるSMAD4の発現の有無又はKLF5の発現を検出し、SMAD4が検出された患者又はKLF5の発現低下若しくは欠失が検出された患者を、請求項1~41のいずれか1項に記載の抗体若しくは当該抗体の機能性断片又は請求項42~48のいずれか1項に記載の医薬組成物による癌の治療の対象者として選別することを含む、癌の治療の対象を選別する方法。
- 癌患者由来の生物学的試料を用い、該生物学的試料中に含まれるSMAD4の発現の有無又はKLF5の発現を検出し、SMAD4が検出された患者又はKLF5の発現低下若しくは欠失が検出された患者に対し、請求項1~41のいずれか1項に記載の抗体若しくは当該抗体の機能性断片又は請求項42~48のいずれか1項に記載の医薬組成物を投与することを含む、癌の治療方法。
- 請求項1~41のいずれか1項に記載の抗体若しくは当該抗体の機能性断片又は請求項42~48のいずれか1項に記載の医薬組成物による癌の治療への応答性を判定するためのキットであって、癌患者由来の生物学的試料中のSMAD4の発現又はKLF5の発現を検出する手段を少なくとも含む、キット。
- 請求項1~41のいずれか1項に記載の抗体又は該抗体の抗原結合断片と他の薬物がコンジュゲートした、抗体薬物複合体。
- 請求項1~41のいずれか1項に記載の抗体の抗原結合断片と、CD147以外の抗原に結合する抗原結合断片を含む、バイスペシフィック抗体。
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KR1020207005574A KR20200032185A (ko) | 2017-07-27 | 2018-07-26 | 항 cd147 항체 |
AU2018307292A AU2018307292A1 (en) | 2017-07-27 | 2018-07-26 | Anti-CD147 antibody |
SG11202000759XA SG11202000759XA (en) | 2017-07-27 | 2018-07-26 | Anti-cd147 antibody |
CN201880049502.XA CN111051513A (zh) | 2017-07-27 | 2018-07-26 | 抗cd147抗体 |
CA3071234A CA3071234A1 (en) | 2017-07-27 | 2018-07-26 | Anti-cd147 antibody |
RU2020108219A RU2785293C2 (ru) | 2017-07-27 | 2018-07-26 | Антитело против cd147 |
MX2020000966A MX2020000966A (es) | 2017-07-27 | 2018-07-26 | Anticuerpo anti-cd147. |
BR112020001657-8A BR112020001657A2 (pt) | 2017-07-27 | 2018-07-26 | Anticorpo contra cd147 humana, fragmento de ligação a antígeno do anticorpo, composição farmacêutica, uso do anticorpo ou do fragmento de ligação a antígeno,polinucleotídeo, vetor de expressão, célula hospedeira, método para produzir o anticorpo ou o fragmento de ligação a antígeno do mesmo, para prognosticar aresponsividade ao tratamento contra o câncer e para selecionar sujeitos para o tratamento contra o câncer, kit para determinar a responsividade ao tratamento contra o câncer, complexo de anticorpo-fármaco, e, anticorpo biespecífico |
EP18838397.0A EP3660155A4 (en) | 2017-07-27 | 2018-07-26 | ANTI-CD147 ANTIBODY |
US16/633,565 US11661451B2 (en) | 2017-07-27 | 2018-07-26 | Anti-CD147 antibody |
JP2019532855A JP7271424B2 (ja) | 2017-07-27 | 2018-07-26 | 抗cd147抗体 |
IL272266A IL272266A (en) | 2017-07-27 | 2020-01-26 | Anti-CD147 antibody |
PH12020500205A PH12020500205A1 (en) | 2017-07-27 | 2020-01-27 | Anti-cd147 antibody |
CONC2020/0001112A CO2020001112A2 (es) | 2017-07-27 | 2020-01-30 | Anticuerpo anti-cd147 |
US17/499,814 US11459387B2 (en) | 2017-07-27 | 2021-10-12 | Anti-CD147 antibody |
US18/315,294 US20230340118A1 (en) | 2017-07-27 | 2023-05-10 | Anti-cd147 antibody |
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Citations (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1988007089A1 (en) | 1987-03-18 | 1988-09-22 | Medical Research Council | Altered antibodies |
WO1990007861A1 (en) | 1988-12-28 | 1990-07-26 | Protein Design Labs, Inc. | CHIMERIC IMMUNOGLOBULINS SPECIFIC FOR p55 TAC PROTEIN OF THE IL-2 RECEPTOR |
US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
WO1992001047A1 (en) | 1990-07-10 | 1992-01-23 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
US5091513A (en) | 1987-05-21 | 1992-02-25 | Creative Biomolecules, Inc. | Biosynthetic antibody binding sites |
WO1992020791A1 (en) | 1990-07-10 | 1992-11-26 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1993006213A1 (en) | 1991-09-23 | 1993-04-01 | Medical Research Council | Production of chimeric antibodies - a combinatorial approach |
WO1993011236A1 (en) | 1991-12-02 | 1993-06-10 | Medical Research Council | Production of anti-self antibodies from antibody segment repertoires and displayed on phage |
WO1993019172A1 (en) | 1992-03-24 | 1993-09-30 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
US5260203A (en) | 1986-09-02 | 1993-11-09 | Enzon, Inc. | Single polypeptide chain binding molecules |
WO1994028027A1 (en) | 1993-06-01 | 1994-12-08 | Arch Development Corporation | Methods and materials for modulation of the immunosuppressive activity and toxicity of monoclonal antibodies |
WO1994029351A2 (en) | 1993-06-16 | 1994-12-22 | Celltech Limited | Antibodies |
WO1995001438A1 (en) | 1993-06-30 | 1995-01-12 | Medical Research Council | Sbp members with a chemical moiety covalently bound within the binding site; production and selection thereof |
WO1995015388A1 (en) | 1993-12-03 | 1995-06-08 | Medical Research Council | Recombinant binding proteins and peptides |
WO1999045031A2 (en) | 1998-03-03 | 1999-09-10 | Abgenix, Inc. | Cd147 binding molecules as therapeutics |
WO1999054342A1 (en) | 1998-04-20 | 1999-10-28 | Pablo Umana | Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity |
WO2000061739A1 (en) | 1999-04-09 | 2000-10-19 | Kyowa Hakko Kogyo Co., Ltd. | Method for controlling the activity of immunologically functional molecule |
WO2002031140A1 (fr) | 2000-10-06 | 2002-04-18 | Kyowa Hakko Kogyo Co., Ltd. | Cellules produisant des compositions d'anticorps |
WO2004061104A2 (en) | 2003-01-07 | 2004-07-22 | Symphogen A/S | Method for manufacturing recombinant polyclonal proteins |
WO2009048072A1 (ja) | 2007-10-11 | 2009-04-16 | Daiichi Sankyo Company, Limited | 破骨細胞関連蛋白質Siglec-15を標的とした抗体 |
WO2010036460A2 (en) | 2008-09-29 | 2010-04-01 | Centocor Ortho Biotech Inc. | Anti-cd147 antibodies, methods, and uses |
WO2010117011A1 (ja) | 2009-04-09 | 2010-10-14 | 第一三共株式会社 | 抗Siglec-15抗体 |
US20150110797A1 (en) * | 2012-04-01 | 2015-04-23 | Michal A. Rahat | Extracellular Matrix Metalloproteinase Inducer (Emmprin) Peptides And Binding Antibodies |
WO2016094566A2 (en) * | 2014-12-10 | 2016-06-16 | Genentech, Inc. | Blood brain barrier receptor antibodies and methods of use |
WO2017061602A1 (ja) | 2015-10-09 | 2017-04-13 | 株式会社ペルセウスプロテオミクス | Cd147分子に特異的に結合する抗体およびその利用 |
JP2017513857A (ja) * | 2014-04-16 | 2017-06-01 | ソレント・セラピューティクス・インコーポレイテッドSorrento Therapeutics, Inc. | Cd147に結合する抗体医薬 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SI2808343T1 (sl) | 2007-12-26 | 2019-10-30 | Xencor Inc | Fc-variante s spremenjeno vezavo na FcRn |
CN104086654B (zh) | 2014-07-04 | 2016-06-08 | 中国人民解放军第四军医大学 | 人源化修饰型抗CD147嵌合抗体HcHAb18及其应用 |
WO2016043577A1 (en) * | 2014-09-16 | 2016-03-24 | Academisch Medisch Centrum | Ig-like molecules binding to bmp4 |
-
2018
- 2018-07-26 JP JP2019532855A patent/JP7271424B2/ja active Active
- 2018-07-26 KR KR1020207005574A patent/KR20200032185A/ko not_active Application Discontinuation
- 2018-07-26 TW TW112137019A patent/TW202402805A/zh unknown
- 2018-07-26 US US16/633,565 patent/US11661451B2/en active Active
- 2018-07-26 EP EP18838397.0A patent/EP3660155A4/en active Pending
- 2018-07-26 CN CN201880049502.XA patent/CN111051513A/zh active Pending
- 2018-07-26 CA CA3071234A patent/CA3071234A1/en active Pending
- 2018-07-26 BR BR112020001657-8A patent/BR112020001657A2/pt unknown
- 2018-07-26 MX MX2020000966A patent/MX2020000966A/es unknown
- 2018-07-26 AU AU2018307292A patent/AU2018307292A1/en active Pending
- 2018-07-26 WO PCT/JP2018/028047 patent/WO2019022187A1/ja active Application Filing
- 2018-07-26 TW TW107125910A patent/TWI818916B/zh active
- 2018-07-26 SG SG11202000759XA patent/SG11202000759XA/en unknown
-
2020
- 2020-01-26 IL IL272266A patent/IL272266A/en unknown
- 2020-01-27 PH PH12020500205A patent/PH12020500205A1/en unknown
- 2020-01-30 CO CONC2020/0001112A patent/CO2020001112A2/es unknown
-
2021
- 2021-10-12 US US17/499,814 patent/US11459387B2/en active Active
-
2023
- 2023-04-26 JP JP2023072046A patent/JP2023093694A/ja active Pending
- 2023-05-10 US US18/315,294 patent/US20230340118A1/en active Pending
Patent Citations (28)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5260203A (en) | 1986-09-02 | 1993-11-09 | Enzon, Inc. | Single polypeptide chain binding molecules |
US5455030A (en) | 1986-09-02 | 1995-10-03 | Enzon Labs, Inc. | Immunotheraphy using single chain polypeptide binding molecules |
WO1988007089A1 (en) | 1987-03-18 | 1988-09-22 | Medical Research Council | Altered antibodies |
US5091513A (en) | 1987-05-21 | 1992-02-25 | Creative Biomolecules, Inc. | Biosynthetic antibody binding sites |
US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
WO1990007861A1 (en) | 1988-12-28 | 1990-07-26 | Protein Design Labs, Inc. | CHIMERIC IMMUNOGLOBULINS SPECIFIC FOR p55 TAC PROTEIN OF THE IL-2 RECEPTOR |
WO1992001047A1 (en) | 1990-07-10 | 1992-01-23 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1992020791A1 (en) | 1990-07-10 | 1992-11-26 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1993006213A1 (en) | 1991-09-23 | 1993-04-01 | Medical Research Council | Production of chimeric antibodies - a combinatorial approach |
WO1993011236A1 (en) | 1991-12-02 | 1993-06-10 | Medical Research Council | Production of anti-self antibodies from antibody segment repertoires and displayed on phage |
WO1993019172A1 (en) | 1992-03-24 | 1993-09-30 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1994028027A1 (en) | 1993-06-01 | 1994-12-08 | Arch Development Corporation | Methods and materials for modulation of the immunosuppressive activity and toxicity of monoclonal antibodies |
WO1994029351A2 (en) | 1993-06-16 | 1994-12-22 | Celltech Limited | Antibodies |
WO1995001438A1 (en) | 1993-06-30 | 1995-01-12 | Medical Research Council | Sbp members with a chemical moiety covalently bound within the binding site; production and selection thereof |
WO1995015388A1 (en) | 1993-12-03 | 1995-06-08 | Medical Research Council | Recombinant binding proteins and peptides |
WO1999045031A2 (en) | 1998-03-03 | 1999-09-10 | Abgenix, Inc. | Cd147 binding molecules as therapeutics |
WO1999054342A1 (en) | 1998-04-20 | 1999-10-28 | Pablo Umana | Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity |
WO2000061739A1 (en) | 1999-04-09 | 2000-10-19 | Kyowa Hakko Kogyo Co., Ltd. | Method for controlling the activity of immunologically functional molecule |
WO2002031140A1 (fr) | 2000-10-06 | 2002-04-18 | Kyowa Hakko Kogyo Co., Ltd. | Cellules produisant des compositions d'anticorps |
WO2004061104A2 (en) | 2003-01-07 | 2004-07-22 | Symphogen A/S | Method for manufacturing recombinant polyclonal proteins |
WO2009048072A1 (ja) | 2007-10-11 | 2009-04-16 | Daiichi Sankyo Company, Limited | 破骨細胞関連蛋白質Siglec-15を標的とした抗体 |
WO2010036460A2 (en) | 2008-09-29 | 2010-04-01 | Centocor Ortho Biotech Inc. | Anti-cd147 antibodies, methods, and uses |
JP2012506369A (ja) * | 2008-09-29 | 2012-03-15 | ヤンセン バイオテツク,インコーポレーテツド | 抗cd147抗体、方法及び使用 |
WO2010117011A1 (ja) | 2009-04-09 | 2010-10-14 | 第一三共株式会社 | 抗Siglec-15抗体 |
US20150110797A1 (en) * | 2012-04-01 | 2015-04-23 | Michal A. Rahat | Extracellular Matrix Metalloproteinase Inducer (Emmprin) Peptides And Binding Antibodies |
JP2017513857A (ja) * | 2014-04-16 | 2017-06-01 | ソレント・セラピューティクス・インコーポレイテッドSorrento Therapeutics, Inc. | Cd147に結合する抗体医薬 |
WO2016094566A2 (en) * | 2014-12-10 | 2016-06-16 | Genentech, Inc. | Blood brain barrier receptor antibodies and methods of use |
WO2017061602A1 (ja) | 2015-10-09 | 2017-04-13 | 株式会社ペルセウスプロテオミクス | Cd147分子に特異的に結合する抗体およびその利用 |
Non-Patent Citations (70)
Title |
---|
"Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY |
"GenBank", Database accession no. NM 001077184. 1 |
ALTSCHUL, STEPHEN F.THOMAS L. MADDENALEJANDRO A. SCHAFFERJINGHUI ZHANGZHENG ZHANGWEBB MILLERDAVID J. LIPMAN: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402, XP002905950, DOI: 10.1093/nar/25.17.3389 |
ANGAL ET AL., MOLECULAR IMMUNOLOGY, vol. 30, no. 1, 1993, pages 105 - 108 |
ANNU. REV. IMMUNOL., vol. 12, 1994, pages 433 - 455 |
BRUGGEMANN ET AL., J. EXP. MED., 1987, pages 1351 - 1361 |
BRUGGEMANN ET AL., J.EXP. MED., 1987, pages 1351 - 1361 |
CARMEN, S. ET AL., BRIEFINGS IN FUNCTIONAL GENOMICS AND PROTEOMICS, vol. 1, no. 2, 2002, pages 189 - 203 |
CHANG, CANCER CHEM.THERA.PHARM., 2007 |
CHEN ET AL., B. M. C., 2014, pages 1471 - 2407 |
CHEN Z ET AL., INT. J. RADIATION ONCOLOGY BIOL. PHYS., 2006, pages 435 - 444 |
CHIAMPANICHAYAKUL ET AL., IMMUNOLOGY, 2006, pages 167 - 178 |
COSTE, I. ET AL., BLOOD, 2001, pages 3984 - 3988 |
DAVID ET AL., CELL |
DAVID ET AL., CELL, vol. 164, no. 5, 2016, pages 1015 - 1030 |
DEEG H ET AL., J. BLOOD, 2001, pages 2052 - 2058 |
DOLL, C. ET AL., CURR. OPIN. ONCOL., vol. 113, 1994, pages 345 - 350 |
FLEGEL, W., TRANSFUSION, 2015, pages S47 - S58 |
GASTON, R., KIDNEY INTERNATIONAL, 1991, pages 141 - 148 |
GIBSON, J.AUST. N. Z., J. MED., 1988, pages 625 - 637 |
GLUZMAN, Y., CELL, vol. 23, 1981, pages 175 - 182 |
GRASS ET AL., BIOSOL. REP, 2016, pages 1 - 16 |
HAHN ET AL., SCIENCE, 1996, pages 350 - 353 |
HAMERS-CASTERMAN C. ET AL., NATURE, vol. 363, no. 6428, 1993, pages 446 - 8 |
HOFFMANN ET AL., J. LEUKOC. BIOL., 2002, pages 847 - 855 |
HU, J. ET AL., J. CELL. MOL. MED., 2010, pages 2132 - 2143 |
HUSTON, J. S. ET AL., PROC. NATL. ACAD. SCI. USA., vol. 85, 1988, pages 5879 - 5883 |
ITO, BLOOD, 2002, pages 3175 - 3182 |
KASINRERK ET AL., IMMUNOLOGY, vol. 96, no. 2, 1999, pages 184 - 192 |
KOCH C ET AL., INTERNATIONAL IMMUNOLOGY, 1999, pages 777 - 786 |
KOCH ET AL., INT. IMMUNOLOGY, 1999, pages 777 - 786 |
KOHLERMILSTEIN, NATURE, vol. 256, 1975, pages 495 - 497 |
KUROIWA, Y. ET AL., NUCL. ACIDS RES., vol. 26, 1998, pages 3447 - 3448 |
LANDRY ET AL., BIOCHEM. CELL BIOL., 1995, pages 703 - 707 |
LEE, H-S ET AL., MOLECULAR IMMUNOLOGY, vol. 36, 1999, pages 61 - 71 |
LEOVONEN ET AL., PLOS ONE, 2013, pages e57474 |
LI, J. HEPATOLOGY, 2015, pages 1378 - 1389 |
LIM ET AL., FEBS LETTERS, 1998, pages 88 - 92 |
LORI BURTON ET AL., PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY, vol. 12, 2007, pages 265 - 273 |
METHODS IN ENZYMOLOGY, vol. 203, 1991, pages 121 - 153 |
MILLSTEIN ET AL., NATURE, vol. 305, 1983, pages 537 - 539 |
MIYAZONO, THE OFFICIAL JOURNAL OF THE JAPAN GERIATRICS SOCIETY, 1999, pages 162 - 166 |
MUYLDEMANS S. ET AL., PROTEIN ENG., vol. 7, no. 9, 1994, pages 1129 - 35 |
NATURE BIOTECHNOLOGY, vol. 23, no. 9, 2005, pages 1137 - 1146 |
NATURE, vol. 321, 1986, pages 522 - 525 |
NUC. ACIDS RES., vol. 35, 2007, pages D301 - D303 |
OVERDIJK ET AL., JOURNAL OF IMMUNOLOGY, 2012, pages 1 - 9 |
PAREKH ET AL., MABS, 2012, pages 310 - 318 |
PROC NATL ACAD SCI USA., vol. 63, no. 1, 1969, pages 78 - 85 |
PROC. NATL. ACAD. SCI. USA., vol. 81, 1984, pages 6851 - 6855 |
QIN, H. ET AL: "CD147-induced cell proliferation is associated with Smad4 signal inhibition", EXPERIMENTAL CELL RESEARCH, vol. 358, no. 2, 3 July 2017 (2017-07-03), pages 279 - 289, XP085186098, DOI: 10.1016/j.yexcr.2017.07.003 * |
QUEEN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 10029 - 10033 |
REDZIC, J., J. MOL. BIOL., 2011, pages 68 - 82 |
SAIKI, R. K. ET AL., SCIENCE, vol. 239, 1988, pages 487 - 49 |
SHIRRMANN, T. ET AL., MABS, vol. 2, no. 1, 2010, pages 1 - 4 |
SIRIWARDENA, D. ET AL., OPHTHALMOLOGY, vol. 109, no. 3, 2002, pages 427 - 431 |
SPRING ET AL., EUR. J. IMMUNOL., 1997, pages 891 - 897 |
SUNTHARALINGAM, G. ET AL., N. ENGL. J. MED., 2006, pages 1018 - 1028 |
TAWARA, T., J. IMMUNOLOGY, 2008, pages 2294 - 2298 |
TOMIZUKA, K. ET AL., NATURE GENETICS, vol. 16, 1997, pages 133 - 143 |
TOMIZUKA, K. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 97, 2000, pages 722 - 727 |
TSUBAKI ET AL., INT. J. BIOL. MACROMOL, 2013, pages 139 - 147 |
URLAUB, G.CHASIN, L. A., PROC. NATL. ACAD. SCI. USA., vol. 77, 1980, pages 4126 - 4220 |
VAFA ET AL., METHODS, vol. 65, 2014, pages 114 - 126 |
VASILAKI ET AL., FASEB JOURNAL, 2010, pages 891 - 905 |
VESSILLIER, S. ET AL., J. IMMUNOLOL. METHODS, 2015, pages 43 - 52 |
WORMSTONE, I. M. ET AL., INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, vol. 43, no. 7, 2002, pages 2301 - 2308 |
XU J ET AL., HEPATOLOGY, 2007, pages 269 - 276 |
YASUTOME ET AL., CLIN. EXP. METASTASIS, 2005, pages 461 - 473 |
ZANG ET AL., CURRENT BIOLOGY, 1997, pages 270 - 276 |
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