WO2014208760A1 - Anticancer monoclonal antibody and method for producing same - Google Patents

Abstract

The present invention addresses the problem of providing a monoclonal antibody which specifically binds to 67LR and is capable of activating the actions of 67LR (it is to be understood that in the specification, a monoclonal antibody includes a chimeric antigen receptor) and a method for producing the same, and as a means for solving the problem, an antibody is produced using a partial peptide of 67LR, for example, 67LR_158-170 peptide sequence: DIAIPCNNKGAHSVG) as an antigen.

Description

Anti-cancer monoclonal antibody and method for producing the same

The present invention relates to a monoclonal antibody, and more particularly to a monoclonal antibody useful for treating cancer, obesity, arteriosclerosis, allergy, inflammation or muscle atrophy, and a method for producing the same.

Cancer is a disease that causes one third of the nation to die, and there is a strong demand for the establishment of appropriate treatment. In leukemia, which is known as a blood cancer, the therapeutic performance has been improved by the induction of differentiation using all-trans retinoic acid (ATRA) and the introduction of molecular targeting drugs for BCR / ABL proteins caused by the Philadelphia chromosome. It's getting on. However, there are many cases in which tumors acquire resistance to these drugs and treatment is difficult, and there are still many problems. Therefore, the development of an anticancer agent having a completely different mechanism of action from the past provides a new option for patients whose conventional anticancer agents have become ineffective. Furthermore, if the dose-limiting toxicity is different, it can be combined with conventional treatment, and a more effective treatment strategy can be formulated.

A 67 kDa laminin receptor protein (67 kDa Laminin Receptor, hereinafter also referred to as 67LR) was originally discovered as a cell membrane protein that binds to laminin, which is a major component of the basement membrane (Non-patent Document 1). In recent studies, 67LR is abnormally enhanced in cancer cells (Non-Patent Document 2), and a strong correlation has been observed between its expression and proliferation, invasion, and metastasis (Non-Patent Documents 3 to 4). 9). In fact, 67LR is highly expressed in all cancer cells such as breast cancer, stomach cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, multiple myeloma, bile duct cancer, colon cancer, uterine cancer, and mesothelioma. (Non-Patent Documents 9-15).

Epigallocatechin gallate (hereinafter also referred to as EGCG), which is one of the main catechins contained in green tea, has been reported to have anticancer activity (Non-patent Document 16), and currently has chronic lymphocytic leukemia patients Is undergoing clinical trials (Non-patent Document 17). The present inventors have clarified that the target molecule on the cell membrane responsible for the anticancer activity of EGCG is a 67 kDa laminin receptor protein (Non-Patent Documents 18 to 21). Furthermore, EGCG is anticancer against cancer cells such as breast cancer, lung cancer, cervical cancer, colon cancer, melanoma, acute myeloid leukemia, multiple myeloma, pancreatic cancer, prostate cancer via 67LR. It has been reported to exert its action (Non-patent Documents 10 to 12, 18 to 21). That is, it has been clarified that EGCG can be a molecular targeting agent for 67LR positive cancer cells. Thus, since EGCG targets 67LR highly expressed in tumors, it has an ideal selective toxicity as a molecular target anticancer agent.
On the other hand, EGCG has been shown to exert anticancer activity by binding to the amino acid sequence of 161-170 of _67LR (67LR _161-170 peptide; IPCNNKGAHS). This peptide sequence is EGCG sensing motif [EGCG Sensing Motif (ESM)] (Non-patent Document 22).

From the above, an antibody that specifically binds to 67LR and can activate the action of 67LR is expected to be useful for diagnosis and treatment of cancer. However, a monoclonal antibody that specifically binds to 67LR and can activate the action of 67LR has not been established so far. Therefore, the present invention provides a monoclonal antibody that specifically binds to 67LR and can activate the action of 67LR (in this specification, when expressed as a monoclonal antibody, it also includes a chimeric antigen receptor) and a method for producing the same There is to do.

The present inventors prepared a hybridoma by using a partial peptide of 67LR as an immunogen, established a monoclonal antibody-producing hybridoma strain that specifically reacts with 67LR, and cultured the hybridoma strain to obtain the following book. Completed the invention.
That is, the present invention relates to the following (1) to (25).
(1) An antibody having anticancer activity by activating 67 kDa laminin receptor protein (hereinafter referred to as 67LR).
(2) An antibody comprising a variable region of EGCG sensing motif (hereinafter referred to as ESM) -8 antibody as an antigen-binding region.
(3) An antibody comprising the variable region of the ESM-16 antibody as an antigen-binding region.
(4) The antibody according to any one of (1) to (3), which is a humanized antibody.
(5) The antibody according to any one of (1) to (3), which is a canine antibody.
(6) The antibody according to any one of (1) to (3), which is a cat antibody.
(7) An antibody comprising a peptide sequence of the complementary determining region (CDR) of the variable region of the ESM-8 antibody.
(8) An antibody comprising a peptide sequence of the complementarity determining region (CDR) of the variable region of the ESM-16 antibody.
(9) In the amino acid sequence of the antibody according to any one of (1) to (8), one or more amino acids are substituted, deleted, added and / or inserted, and (1) to (8) An antibody having an activity equivalent to that of the antibody according to any one of the above.
(10) A chimeric antigen receptor comprising the peptide sequence of the variable region of the ESM-8 antibody.
(11) A chimeric antigen receptor comprising the peptide sequence of the variable region of the ESM-16 antibody.
(12) In the amino acid sequence of the antibody according to any one of (1) to (9), one or more amino acids are substituted, deleted, added and / or inserted, and (1) to (9) A chimeric antigen receptor having an activity equivalent to that of the antibody according to any one of the above.
(13) Green tea catechin EGCG has any of the anti-cancer activity, anti-obesity, anti-arteriosclerotic action, anti-allergy, anti-inflammatory or muscular atrophy inhibition physiological activity expressed through 67LR, (1) to (9 The antibody according to any one of 1).
(14) The antibody according to any one of (1) to (9), which has an apoptosis-inducing activity by activating acidic sphingomyelinase that directly participates in apoptosis induction of green tea catechin EGCG.
(15) An anticancer agent comprising a combination of the antibody according to any one of (1) to (9) and any one of a PDE3 inhibitor, a PDE5 inhibitor, a Shpk1 inhibitor, or a gemcitabine.
(16) A diagnostic antibody for discriminating an adaptor of the antibody according to any one of (1) to (9).
(17) An antibody in which a radioisotope, a fluorescent agent, an enzyme, a cytotoxin, or the like is bound to the antibody according to any one of (1) to (9).
(18) A method for obtaining an antibody using a peptide sequence on the 67LR molecule to which green tea catechin EGCG binds as an immunizing antigen.
(19) A method for obtaining an antibody exhibiting anticancer activity using a peptide sequence on the 67LR molecule to which green tea catechin EGCG binds as an immune antigen.
(20) Anti-cancer activity, anti-obesity, anti-arteriosclerosis action, anti-allergy, anti-inflammatory or muscle atrophy expressed by green tea catechin EGCG via 67LR with the peptide sequence on 67LR molecule bound by green tea catechin EGCG A screening method for an antibody having any of the inhibitory physiological activities.
(21) When combined with a PDE3 inhibitor, PDE5 inhibitor, Shpk1 inhibitor, or gemcitabine, any of anti-cancer activity, anti-obesity, anti-arteriosclerotic effect, anti-allergy, anti-inflammatory, or muscle atrophy inhibiting physiological activity is enhanced Antibody screening method.
(22) An antibody obtained by the method according to any one of (18) to (21).
(23) A method for treating cancer, obesity, arteriosclerosis, allergy, inflammation or muscle atrophy, comprising administering the antibody according to any one of (1) to (9) to a subject.
(24) The gene of ESM-8 antibody is a group consisting of a human-derived cell line, a hamster-derived cell line, a mouse-derived cell line, an insect-derived cell line, a sugar chain biosynthesis mutant cell line of these cell lines, and Escherichia coli and yeast. The antibody according to any one of (1) to (9), which comprises introducing into a selected host cell, culturing the host cell to express the antibody, and collecting the expressed antibody. how to.
(25) The gene of ESM-16 antibody is derived from a human cell line, a hamster cell line, a mouse cell line, an insect cell line, a sugar chain biosynthesis mutant cell line of these cell lines, and a group consisting of E. coli and yeast. The antibody according to any one of (1) to (9), which comprises introducing into a selected host cell, culturing the host cell to express the antibody, and collecting the expressed antibody. how to.

According to the present invention, an antibody that specifically binds to a 67 kDa laminin receptor protein can be provided.

FIG. 1 (a) is a graph showing antibody titers against 67LR _161-170 peptide in serum using sera collected from mice immunized with KLH, KLH, and recombinant 67LR to which 67LR _158-170 peptide is bound as antigens. It is. The horizontal axis shows the immunogen, and the vertical axis shows the absorbance obtained by the ELISA method. FIG. 1 (b) shows the result of measuring the antibody titer by ELISA method for the binding activity of the antibody produced by each hybridoma to 67LR _161-170 peptide after cloning the obtained hybridoma by limiting dilution method. Hybridoma clone no. The absorbance obtained by the ELISA method is shown on the vertical axis. FIG. 2 shows the results of measurement of anti-67LR _161-170 peptide antibody in the culture supernatant obtained from each hybridoma clone. Hybridoma clone no. The absorbance obtained by the ELISA method is shown on the vertical axis. FIG. 3 is a graph showing the results of measuring the binding of the ESM-8 antibody to the 67LR _161-170 peptide and the control peptide by ELISA. The horizontal axis represents the antibody concentration, and the vertical axis represents the absorbance obtained by the ELISA method. FIG. 4 is a graph showing the results of measuring the binding of the ESM-16 antibody to the 67LR _161-170 peptide and the control peptide by ELISA. The horizontal axis represents the antibody concentration, and the vertical axis represents the absorbance obtained by the ELISA method. FIG. 5 shows the DNA sequence of the ESM-8 antibody variable region. Leader sequences are in lower case, CDRs are in bold, and FRs are underlined. FIG. 6 shows the DNA sequence of the ESM-16 antibody variable region. Leader sequences are in lower case, CDRs are in bold, and FRs are underlined. FIG. 7 is a graph showing the results of examining the cell growth inhibitory activity of ESM-8 antibody against human acute myeloid leukemia cell line HL60. The horizontal axis indicates the sample type and the concentration used, and the vertical axis indicates the number of living cells. FIG. 8 shows the results of examining the binding activity of ESM-8 antibody to the cell surface of human acute myeloid leukemia cell line HL60. The horizontal axis indicates the light intensity, and the vertical axis indicates the number of cells. FIG. 9 shows the results of examining the binding activity of the ESM-16 antibody to the cell surface of human multiple myeloma cell line U266. The horizontal axis indicates the light intensity, and the vertical axis indicates the number of cells. FIG. 10 (a) shows the results of measuring the sphingomyelinase activity of the ESM-8 antibody. FIG. 10 (a) is a photograph showing the results of TLC, and FIG. 10 (b) shows the relative sphingomyelinase activity. FIG. 11 shows the results of testing whether the EMS-8 antibody exhibits a cytostatic effect on the human acute myeloid leukemia cell line HL60. The horizontal axis indicates the combination of samples, and the vertical axis indicates the relative number of living cells. FIG. 12 shows the results of examining the cell growth inhibitory activity of the recombinant ESM-8 antibody against human acute myeloid leukemia cell line HL60, human pancreatic cancer cell line PANC-1 and human mesothelioma cell line MESO-4 It is a graph. The horizontal axis indicates the type of sample and the concentration used, and the vertical axis indicates the number of living cells. FIG. 13 shows the results of measuring the sphingomyelinase activity of the recombinant ESM-8 antibody. FIG. 14 shows evaluation of the binding activity of recombinant ESM-8 antibody to the surface of acute myeloid leukemia cell line HL60 cells. FIG. 15 shows the anticancer activity of the recombinant ESM-8 antibody when combined with each inhibitor of PDE3 inhibitor, PDE5 inhibitor and sphingosine kinase. In the figure, + indicates that each inhibitor is used, and-indicates that each inhibitor is not used. The vertical axis shows the relative number of living cells. FIG. 16 shows the anticancer activity of the recombinant ESM-8 antibody in combination with gemcitabine. In the figure, + indicates that gemcitabine is used, and − indicates that gemcitabine is not used. The vertical axis shows the relative number of living cells. FIG. 17 is a graph showing the cell proliferation activity of EMS-16 antibody against human multiple myeloma cell U266, mouse melanoma cell line B16 and human pancreatic cancer cell line PANC-1. The number of viable cells is shown on the vertical axis.

In the present invention, the 67 kDa laminin receptor protein (67LR) is a protein discovered as a cell membrane protein that binds to laminin, which is a major component of the basement membrane, specifically the amino acid represented by SEQ ID NO: 1. It means a protein having a sequence. In recent studies, 67LR is abnormally enhanced in cancer cells, and a strong correlation has been observed between its expression and proliferation, invasion, and metastasis. There have been many reports that 67LR is hardly expressed in normal lymphocytes and is highly expressed in leukemia cells. On the other hand, epigallocatechin gallate (hereinafter also referred to as EGCG), which is one of the main catechins contained in green tea, has been reported to have an anticancer activity, and is currently clinically used in patients with chronic lymphocytic leukemia. A test is being conducted. The present inventors have clarified that the target molecule on the cell membrane responsible for the anticancer activity of EGCG is a 67 kDa laminin receptor protein.

The monoclonal antibody of the present invention may be any antibody that specifically binds to 67LR, but preferably recognizes a peptide sequence consisting of amino acids 161 to 170 from the N-terminus of 67LR. . In addition, the monoclonal antibody of the present invention can be established by a specific known production method.

As described above, since the target molecule on the cell membrane responsible for the anticancer activity of EGCG contained in green tea catechin has been clarified to be a 67 kDa laminin receptor protein, the peptide sequence on 67LR to which EGCG binds is represented. It can be used as an immunizing antigen to obtain antibodies. In addition, the antibody obtained as described later exhibits anticancer activity. The present invention uses a peptide sequence on a 67 kDa laminin receptor protein molecule to which epigallocatechin gallate in green tea catechin binds as an immunizing antigen, and epigallocatechin gallate in green tea catechin is mediated through a 67 kDa laminin receptor protein. A method for screening an antibody having one or more physiological activities selected from the group consisting of anti-cancer activity, anti-obesity action, anti-arteriosclerosis action, anti-allergic action, anti-inflammatory action and muscle atrophy inhibiting action To do. Specifically, antibodies can be screened by the following method.

Antigens used to produce anti-67LR monoclonal antibodies include cells that produce 67LR or cell fractions thereof, or host cells transformed with DNA encoding 67LR, ie prokaryotic host cells such as E. coli A full-length or partial fragment of cDNA encoding 67LR into a eukaryotic host cell such as an insect cell or a mammalian cell using a known method, and expressed or purified as it is or as a fusion protein; A 67LR partial peptide synthesized using a peptide synthesizer can be used. As the partial peptide, a peptide consisting of a sequence of 3 or more residues in the peptide sequence consisting of amino acids 161 to 170 from the N-terminal of 67LR can be used, preferably 161 to 170th from the N-terminal of 67LR. A peptide comprising a peptide sequence consisting of the amino acids 161 to 170 from the N-terminal of 67LR, more preferably a peptide comprising a peptide sequence consisting of the amino acid. Further, it may be a peptide having a total length of 30 residues or less including a peptide sequence consisting of amino acids 161 to 170 from the N-terminal of 67LR.

The monoclonal antibody of the present invention consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence constituting the monoclonal antibody, and is substantially the same biology as the monoclonal antibody. Antibodies that have specific activity are also encompassed by the present invention. The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited. However, deletion, substitution, insertion or addition is performed by a well-known technique such as site-directed mutagenesis. It is a number that can be done. For example, the number is 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.

The deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence constituting the monoclonal antibody means the following. It means that there is a deletion, substitution, insertion or addition of one or more amino acid residues in any and one or more amino acid sequences in the same sequence. Deletions, substitutions, insertions or additions may occur simultaneously.

Monoclonal antibodies of the present invention include monoclonal antibody ESM-8 and monoclonal antibody ESM-16. Further, the monoclonal antibody of the present invention may contain a peptide sequence of a part or all of the complementarity determining region of the variable region of the monoclonal antibody ESM-8 or ESM-16. The complementarity determining region of the variable region means three regions, CDR1, CDR2, and CDR3, which are sites that exist in the variable region of an antibody and directly complementarily bind to an antigen.
Monoclonal antibodies ESM-8 and ESM-16 can be prepared by the methods described in the Examples described later.
Specific examples of the monoclonal antibody of the present invention include the following monoclonal antibodies.
(A) The amino acid sequence of the heavy chain variable region includes the amino acid sequence represented by SEQ ID NO: 6 or 22, and / or the amino acid sequence of the light chain variable region includes the amino acid sequence represented by SEQ ID NO: 8 or 24 Monoclonal antibody (b) Monoclonal antibody in which the amino acid sequence of the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 6 and / or the amino acid sequence of the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 8 (C) a monoclonal antibody in which the amino acid sequence of the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 22 and / or the amino acid sequence of the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 24 (d ) The amino acid sequence of CDR of heavy chain variable region includes the amino acid sequence represented by SEQ ID NO: 10, 12, 14 or SEQ ID NO: 26, 28, 30 And / or monoclonal antibody comprising the amino acid sequence of CDR of light chain variable region represented by SEQ ID NO: 16, 18, 20 or SEQ ID NO: 32, 34, 36 (e) amino acid of CDR of heavy chain variable region A monoclonal antibody comprising an amino acid sequence represented by SEQ ID NO: 10, 12, 14 and / or a CDR amino acid sequence of the light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 16, 18, 20 ( f) The amino acid sequence of the CDR of the heavy chain variable region includes the amino acid sequence represented by SEQ ID NO: 26, 28, 30 and / or the amino acid sequence of the CDR of the light chain variable region is SEQ ID NO: 32, 34, 36 The present invention relates to a monoclonal antibody ESM-8 or a monoclonal antibody ESM-16. Also provides chimeric antigen receptor comprise a peptide sequence of the variable region.
In the present specification, a chimeric antigen receptor (CAR) is a genetically engineered linkage of a variable region of a light chain and a heavy chain of a monoclonal antibody specific for a tumor antigen and a T cell receptor ζ chain. Means a chimeric protein. Today, T cell receptor gene-modified immune cell therapy for cancer has attracted attention, and in this therapy, a chimeric antigen receptor (CAR) that has been artificially designed to recognize cancer cell surface molecules. It is characterized by a high antitumor effect of T cells into which a gene has been introduced and expressed, and versatility independent of HLA (Human Leukocyte Antigen). The variable regions of ESM-8 and ESM-16, which are monoclonal antibodies against a kind of tumor antigen 67LR, can be used to create a novel chimeric antigen receptor CAR.

The monoclonal antibody of the present invention includes antibodies produced by genetic recombination such as human chimeric antibodies, humanized antibodies, human antibodies or antibody fragments thereof. Recombinant antibodies are particularly preferred for use as therapeutic agents because they have the characteristics of monoclonal antibodies and reduce the heteroantigenicity of humans. These modified antibodies can be produced using known methods. The chimeric antibody includes an antibody in which the variable region and the constant region of the antibody are heterologous to each other. For example, mammals other than humans, for example, the heavy chain of a mouse antibody, the variable region of a light chain and the heavy chain of a human antibody, An antibody consisting of the constant region of the chain can be mentioned. Such an antibody can be obtained by ligating DNA encoding the variable region of a mouse antibody with DNA encoding the constant region of a human antibody, incorporating it into an expression vector, introducing it into a host, and producing it.

In addition, when a monoclonal antibody of the present invention is administered to dogs and cats, the monoclonal antibody of the present invention is preferably a canine antibody, a canine antibody, a cat antibody, or a cat antibody.
“Inuization” is defined as a method of transferring non-canine antigen binding information from a donor antibody to a weaker immunogenic canine antibody acceptor, producing a treatment useful as a therapy in a dog.

“Catification” is defined as a method of transferring non-cat antigen binding information from a donor antibody to a weaker immunogenic cat antibody acceptor to produce a treatment useful as a therapy in a cat.
Known methods can be used for producing canine antibodies, canine antibodies, feline antibodies, and feline antibodies.

The monoclonal antibodies of the invention can be labeled for detection. Monoclonal antibodies can be labeled by binding radioisotopes, fluorescent agents or enzymes. Examples of the radioisotope include ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I. Examples of the fluorescent agent include fluorescein and rhodamine. Examples of the enzyme include various enzymes well known in the art such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, xanthine oxidase, glucose oxidase or other sugar oxidase or luciferase.
Further, the monoclonal antibody of the present invention may be conjugated with a cytotoxin. Thus, by binding with a cytotoxin, it becomes possible to direct a toxic agent to the cell expressing the antigen of the monoclonal antibody of the present invention. Examples of the cytotoxin include Pseudomonas exogenous toxin.
There is no limitation on the method for binding the radioisotope, the fluorescent agent, the enzyme or the cytotoxin to the monoclonal antibody, and it can be carried out by a conventionally known method.

Next, the medicament of the present invention will be described.
The medicament of the present invention contains the monoclonal antibody of the present invention as an active ingredient.
The medicament of the present invention can treat various diseases involving 67LR.
Diseases involving 67LR include cancer, obesity, arteriosclerosis, allergy, inflammation and muscle atrophy. As cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, multiple myeloma, bile duct cancer, colon cancer, cervical cancer and 67LR high expression like these cancers Mesothelioma.

The monoclonal antibody of the present invention is a melanoma cell line B16 cell in which EGCG exerts an anticancer action via 67LR, acute myeloid leukemia cell line HL60, pancreatic cancer cell line PANC-1, mesothelioma cell line MRSO- 4 exhibits anti-cancer activity. In addition, EGCG activates acidic sphingomyelinase to cancer cells and exhibits anticancer activity, but the monoclonal antibody of the present invention activates acidic sphingomyelinase to cancer cells in the same manner as EGCG. Furthermore, the monoclonal antibody of the present invention exhibits an anticancer activity in a 67LR-dependent manner, like EGCG. This shows that the monoclonal antibody of the present invention functions as a 67LR agonist like EGCG, and breast cancer, stomach cancer, pancreatic cancer, which are cancer types in which EGCG exerts an anticancer action via 67LR. ESM-8 for prostate cancer, acute myeloid leukemia, multiple myeloma, bile duct cancer, colon cancer, cervical cancer and mesothelioma that expresses 67LR as well as these cancers It has been shown that antibodies and ESM-16 antibodies can be anticancer agents (Non-Patent Documents 9 to 15).

The medicament of the present invention may further contain a PDE3 inhibitor, a PDE5 inhibitor, a sphingosine kinase inhibitor, or gemcitabine. PDE3 inhibitors include, for example, trekinsin, cilostazol (6- [4- (1-cyclohexyl-1H-tetrazol-5-yl) butoxy] -3,4-dihydro-2 (1H) -quinolone, or 6- [4- (1-cyclohexyl-1H-tetrazol-5-yl) butoxy] -3,4-dihydro-2 (1H) -quinolinone) and the like. May be used. Examples of the PDE5 inhibitor include sildenafil, vardenafil, tadalafil, udenafil and the like. Sphingosine kinase inhibitors include, for example, saphingol (L-threo-dihydrosphingosine), N, N-dimethylsphingosine, trimethylsphingosine and analogs and derivatives of sphingosine such as dihydrosphingosine and myriocin.

The medicament of the present invention can be administered alone, but usually mixed together with one or more pharmacologically acceptable carriers, and any well known in the technical field of pharmaceutics It is preferably provided as a pharmaceutical preparation produced by the method.

It is preferable to use the most effective route for treatment. Examples of the administration route include oral administration and parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous. In the case of protein preparations, intravenous administration is preferred.

Examples of administration forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, and tapes.

Examples of preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.

Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p- Preservatives such as hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint can be used as additives.

Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl A binder such as alcohol, hydroxypropylcellulose, and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin can be used as additives.

Examples of preparations suitable for parenteral administration include injections, suppositories, and sprays.

The injection is prepared using, for example, a carrier made of a salt solution, a glucose solution, or a mixture of both.

The suppository is prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.

The spray is prepared using a carrier that does not irritate the drug itself or the recipient's oral cavity and airway mucosa, and that disperses the drug as fine particles to facilitate absorption.

Specific examples of the carrier include lactose and glycerin. Depending on the properties of the medicine and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.

The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, weight, etc., but it is usually preferably 1 to 30 mg / kg as the amount of antibody per adult. The drug used in combination with the antibody is preferably at the same dose or less than the dose when used alone clinically.

It has been reported that by using EGCG as a carrier for a drug delivery system (DDS), it is possible to accumulate cancer-specific drugs targeting 67LR (Non-patent Document 24). Since the monoclonal antibody of the present invention specifically binds to 67LR, the medicament of the present invention can be used for breast cancer, stomach cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, multiple myeloma, bile duct cancer, colon cancer. , Cervical, mesothelioma cancers that highly express 67LR, tumors targeting 67LR and carriers for accumulating radiation sources, radioisotopes, fluorescent agents, enzymes, cytotoxins, anticancer agents, etc. Diagnosis, sensitivity evaluation, and armed antibody.

EGCG not only works directly on cancer cells to exert an anticancer effect, but also activates tumor immunity via 67LR to develop an anticancer effect (Non-patent Document 25). Therefore, since the monoclonal antibody of the present invention has a property as a 67LR ligand, it is expected to be applied as a tumor immunity activator that exhibits anticancer activity against a wide range of cancers by activating tumor immunity in the same manner as EGCG. it can. That is, the medicament of the present invention can be expected to be applied as a tumor immune activator.
Since the monoclonal antibody of the present invention activates acidic sphingomyelinase that is directly involved in the induction of apoptosis of EGCG, it is considered to have an apoptosis-inducing activity. Therefore, the medicament of the present invention can induce apoptosis in cancer cells.

EGCG not only has an anti-cancer effect but also an inflammatory reaction inhibitory effect (Non-patent document 26), anti-allergic effect (Non-patent document 27), arteriosclerosis / thromboprophylaxis (Non-patent document 28), and muscle atrophy suppression Because of its action (Non-Patent Document 29), a medicament containing the monoclonal antibody of the present invention showing the same activity as EGCG by binding to the EGCG binding site on the 67LR molecule is an antiallergic agent, antiarteriosclerosis / thrombosis It can be expected to be used as a preventive agent and a muscle atrophy inhibitor. Moreover, in fat cells, EGCG inhibits glucose uptake caused by insulin via 67LR (Non-patent Document 30), and inhibits release of histamine, which is an adipocyte hypertrophy inducer, via 67LR ( Non-patent document 31) can be expected to be used as a pharmaceutical anti-obesity drug containing the monoclonal antibody of the present invention.

The monoclonal antibody of the present invention and the medicament of the present invention can also be used as a diagnostic antibody for discriminating the appropriate person who applies the monoclonal antibody of the present invention.

Since the monoclonal antibody of the present invention specifically binds to 67LR, it can be used for immunological detection, detection and quantification of 67LR.
Since the monoclonal antibody of the present invention specifically binds to 67LR, it can be used for discriminating who can apply the monoclonal antibody of the present invention. The medicament of the present invention can be used for treatment of a disease involving 67LR, but it can be determined using the monoclonal antibody of the present invention whether or not the disease to be treated is a disease associated with 67LR. is there.

As described above, the monoclonal antibody of the present invention and the pharmaceutical agent of the present invention are useful for the treatment of diseases involving 67LR, such as cancer, obesity, arteriosclerosis, allergy, inflammation and muscle atrophy. Accordingly, the present invention provides a method for treating cancer, obesity, arteriosclerosis, allergy, inflammation or muscle atrophy, comprising administering to a subject a monoclonal antibody of the present invention.

The present invention will be described more specifically by the following examples, but the examples are illustrative of the present invention and do not limit the scope of the present invention.

[Example 1]
Immunization using recombinant 67LR (r-hLR _1-295 ) and 67LR _158-170 (1) Preparation and immunization of immunogens Kanamycin used in the following examples is from Wako Pure Chemical Industries, Ltd., IPTG is Funakoshi Corporation Further, imidazole was purchased from Nacalai Tesque Co., Ltd. and HiTrap Chelating HP column was purchased from GE Healthcare (UK). The ultrasonic disruption of E. coli was performed by Handy Sonic (Tomy Seiko Co., Ltd.). The LB liquid medium was prepared by dissolving Tryptone (BD, USA) 10 g / L, Yeast Extract (BD, USA) 5 g / L, and NaCl (Wako Pure Chemical Industries, Ltd.) 10 g / L in ultrapure water. PBS (pH 7.4) is NaCl (Wako Pure Chemical Industries, Ltd.) 8.0 g / L, KCl (Nacalai Tesque Co., Ltd.) 0.2 g / L, Na ₂ HPO ₄ (Wako Pure Chemical Industries, Ltd.) 1.15 g / L, KH ₂ PO ₄ (Nacalai Tesque Co., Ltd.) 0.2 g / L was dissolved in ultrapure water.

As a control for the ESM peptide, recombinant 67LR (r-hLR _1-295 ) was prepared by the following procedure. One Escherichia coli (pET30a-h67LR-introduced BL21) was inoculated into 18 mL of LB liquid medium containing kanamycin (final concentration: 50 μg / mL) and cultured overnight at 37 ° C. with shaking. After culturing, the bacterial cell culture solution was added to two 250 mL LB liquid media and cultured with shaking at 37 ° C. Thereafter, IPTG (final concentration: 1 mM) was added, and cultured with shaking at 37 ° C. for 22 hours to induce the T7 promoter to express r-hLR _1-295 . E. coli was pelleted by centrifugation at 5000 rpm for 15 minutes, washed with PBS, and the resulting pellet was stored frozen at -20 ° C. The cryopreserved pellet was suspended in a crushing buffer (pH 8.0 Tris-HCl / NaCl) and then sonicated. Thereafter, the mixture was centrifuged at 15000 rpm for 10 minutes, and the supernatant was collected and used as a sample.

The sample obtained as described above was applied to a HiTrap Chelating HP column (5 mL). Column adsorption washing was performed with 10 mM imidazole (E0), and elution was performed using 20, 40, 60, 100, 300, 500 mM imidazole-containing buffer. 5 mL of each elution fraction was collected, and the collected sample was dialyzed with the same solvent as the KLH-MB-ESM peptide complex solution at 4 ° C. for about 16 hours. At this time, the solution was exchanged twice in the middle. Samples were collected in siliconized tubes and stored frozen at -20 ° C.
Hemocyanin (keyhole limpet hemocyanin; KLH) was purchased from Wako Pure Chemical Industries. m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was purchased from PIERCE, and dimethylformamide (DMF) was purchased from Sigma (St. Louis, MO). 10 mM sodium phosphate buffer (pH 7.2) was mixed with 0.2 M NaH ₂ PO ₄ solution and 0.2 M Na ₂ HPO ₄ solution at a ratio of 7:18, and the mixture was diluted 20 times with ultrapure water. A product prepared by dilution and autoclaved was used. 50 mM sodium phosphate buffer (pH 6.0) was mixed with 0.2 M NaH ₂ PO ₄ solution and 0.2 M Na ₂ HPO ₄ solution at a ratio of 877: 123, and the mixture was quadrupled with ultrapure water. A product prepared by dilution and autoclaved was used. NaH ₂ PO ₄ and Na ₂ HPO ₄ were purchased from Wako Pure Chemical Industries, Ltd. The 67LR _158-170 peptide was synthesized by a conventional method.

67LR _158-170 peptide sequence: DIAIPCNNKGAHSVG) (SEQ ID NO: 2) was combined with KLH by the MBS method to obtain an immunogen. That is, 20 mg of KLH was dissolved in 1 mL of 10 mM sodium phosphate buffer (pH 7.2), and 3.3 mg of MBS dissolved in 11 μmL of dimethylformamide (DMF) was added to this solution and reacted at room temperature for 30 minutes. . Subsequently, the reaction solution was centrifuged (18000 × g, 5 minutes, 4 ° C.), and the supernatant was collected. The supernatant sample was dialyzed with 50 mM sodium phosphate buffer (pH 6.0) at 4 ° C. for about 16 hours to separate KLH-MB and free MBS. At this time, the buffer was exchanged twice in the middle, and then the sample was collected in a siliconized tube. 1 mg of 67LR _158-170 peptide is dissolved in 500 μmL of sterilized ultrapure water, this solution is mixed with 100 μL of KLH-MB recovered above, and the pH is adjusted to 7.3-7 with a sterilized 0.2 M Na ₂ HPO ₄ solution. .5. Nitrogen gas was blown into the mixed solution, and the mixture was shaken at room temperature for 3 hours to be reacted. Thereafter, the reaction solution was dialyzed with a solution having the same composition and pH as the reaction solution at 4 ° C. for about 16 hours. At this time, the solution was exchanged twice in the middle. Samples were collected in siliconized tubes and stored frozen at -20 ° C.
Six-week-old BALB / c mice were purchased from Kudo Co., Ltd., and reared in a SPF (specific-pathogen free) environment (bred room temperature 20 ± 1 ° C., relative humidity 60 ± 5%). Illumination was adjusted with a white fluorescent lamp mounted on the ceiling, and the period was 12 hours. The illumination time was set to 12 hours from 8:00 to 20:00. As an initial immunization, a recombinant 67LR (r-hLR _1-295 ) or KLH-MB-67LR _158-170 solution as an antigen and an equal amount of a complete adjuvant (Sigma) are vigorously mixed to prepare an emulsion. Each mouse was intraperitoneally administered with 0.2 mL of emulsion. For booster immunization, an incomplete adjuvant (Sigma) was used, and an emulsion was prepared and administered in the same manner as the first time. Immunization was performed at intervals of 2 weeks.

(2) Preparation of anti-67LR _161-170 peptide monoclonal antibody-producing hybridoma 50% polyethylene glycol solution was purchased from Sigma (St. Louis, MO), and thymidine, hypoxanthine, and aminopterin in HAT medium were purchased from Wako Pure Chemical Industries, Ltd. . The mouse myeloma cell line Sp2 / O-Ag14 used for cell fusion was cultured at 37 ° C. in an E-RDF medium (Kyokuto Pharmaceutical Co., Ltd.) supplemented with 5% fetal calf serum (FCS) (Bio Source International, Camarillo, Calif.). And passaged and maintained under steam saturated 5% CO ₂ conditions. 1.125 g of NaHCO ₃ (Wako Pure Chemical Industries, Ltd.) was added to 1 L of E-RDF medium. 8-Azaguanine dissolved in dimethyl sulfoxide (Nacalai Tesque, Inc.) at 500 times concentration was prepared and added every other day. Cells were maintained in culture in the logarithmic growth phase. Sp2 / O-Ag14 was subcultured for 1 week in the presence of 20 μmg / mL of 8-azaguanine (Tokyo Chemical Industry Co., Ltd.), and only the 8-azaguanine resistant strain, that is, the HGPRT-deficient strain was used for cell fusion. The hybridoma is cultured in an E-RDF medium containing 15% FCS, 9.3 × 10 ⁻⁵ M hypoxanthine at 37 ° C., 5% CO ₂ until cell scale is increased from 96 well plate to 24 well plate. The measurement was performed under the same conditions, and after scaling up to 24 well plate, the same procedure was performed with an FCS concentration of 5%.
Three days after the final immunization, one of three immunized mice was sacrificed by blood sampling of the abdominal aorta under isoflurane (Merck Pharmaceutical, Germany, Darmstadt) anesthesia, and the spleen was removed. After excision, lymphocytes were prepared, suspended in 10 mL of E-RDF medium, and the number of viable cells was examined by trypan blue method. The Sp2 / O suspension with 1/10 cell count relative to the total number of spleen cells was added to the spleen cell suspension, mixed, and centrifuged at 300 × g for 5 minutes. The supernatant was completely removed, the cells were loosened by tapping and the temperature was returned to room temperature. 1 mL of a 50% strength polyethylene glycol (PEG) solution was taken into a Pasteur pipette and mixed with the cells for 1 minute. At this time, the pipette tip was gently and surely stirred so that the cells were uniformly dispersed in PEG. Immediately after PEG addition, the whole solution was tapped once, and then 9 mL of ERDF medium was slowly added at a rate of 1 mL / 30 seconds. At this time, the liquid was mixed using the tip of a pipette. After centrifugation at 300 × g for 5 minutes to completely remove the supernatant, the density of splenocytes was 4.6 × 10 ⁶ cells in E-RDF medium containing 15% FCS and 9.3 × 10 ⁻⁵ M hypoxanthine. / ML to float. This cell suspension was dispensed onto six 96 well plates at a rate of 100 μL / well. After culturing at 37 ° C. for 12 hours in the presence of 5% CO ₂ , E-RDF medium containing 15% FCS, 9.3 × 10 ⁻⁵ M hypoxanthine, 8.0 × 10 ⁻⁷ M aminopterin was added to a 96-well plate. The culture was dispensed at a rate of 100 μL / well, and the culture conditions were adjusted to HAT. Static culture was performed at 37 ° C. in the presence of 5% CO ₂ .

(3) Screening of hybridoma producing anti-67LR _161-170 peptide monoclonal antibody
The hybridoma cell cells selected by the HAT medium were seeded on a 96-well plate so as to be 0.2 cells / well with 15% FCS ERDF, and cloning was performed.
A solution obtained by _diluting 67LR _161-170 peptide (IPCNNKGAHS) to a concentration of 10 μg / mL with a solid phase antigen dilution solution was added to ELISA plate Nunc-Immuno plate, Thermo Scientific Nunc, Denmark, and 100 μl was added at 37 ° C. Let stand for hours. As a solid phase antigen diluted solution, NaHCO ₃ (Nacalai Tesque) was prepared to 4.2 g / L with ultrapure water and 50 mM NaHCO ₃ was mixed with Na ₂ CO ₃ (Nacalai Tesque) with ultrapure water. A 50 mM carbonate buffer prepared by mixing 50 mM Na ₂ CO ₃ prepared to 65 g / 500 mL until pH 9.6 was used. After washing the ELISA plate, 50 μL of the culture supernatant of each hybridoma was added and allowed to stand at 37 ° C. for 1 hour. For washing the ELISA plate, PBS (TPBS) to which 0.05% Tween 20 (Nacalai Tesque, Inc.) was added was used. After washing the ELISA plate, 100 μL of a 10,000-fold diluted solution of an HRP-labeled anti-mouse IgG antibody (Zymed Laboratories, Inc, San Frascisco, Calif.) Or a HRP-labeled anti-mouse IgM antibody was added at 100 μL, and 1 at 37 ° C. Let stand for hours. After washing the ELISA plate, 100 μl of chromogenic substrate solution was added. The chromogenic substrate was 0.2 M citrate buffer (pH 4.0) containing 0.006% H ₂ O ₂ immediately before use: ultrapure water: 6 mg / mL 2,2-anizo-bis (3′-ethylbenzonin-6) -Sulphonic acid) Diammonium salt (ABTS) solution = 10: 9: 1. For measuring the absorbance, ImmunoMini NJ-2300 manufactured by Tosoh Corporation was used, and the absorbance at 405 nm was measured.

The measurement results are shown in FIGS. FIG. 1 (a) is a graph showing antibody titers against 67LR _161-170 peptide in serum using sera collected from mice immunized with KLH, KLH, and recombinant 67LR to which 67LR _158-170 peptide is bound as antigens. It is. As apparent from FIG. 1 _(a), although the mice immunized with KLH conjugated with _{67LR 158-170} peptide induced the production of antibodies that bind to _{67LR 161-170} peptide, the KLH or recombinant 67LR as antigen No antibody production against the 67LR _161-170 peptide was observed in the immunized mice.
Next, the antibody of the hybridoma prepared by fusing the mouse spleen in which the production of anti-67LR _161-170 peptide antibody was induced by immunization with 67LR _158-170 peptide as an antigen and SP2 cells was evaluated. FIG. 1 (b) shows the result of measuring the antibody titer by ELISA method for the binding activity of the antibody produced by each hybridoma to the 67LR _161-170 peptide after cloning of the obtained hybridoma by the limiting dilution method. As is clear from FIG. 1 (b), a plurality of hybridomas producing antibodies against the 67LR _161-170 peptide were obtained. Among them, antibodies showing strong reactivity to ESM peptides were obtained in the culture supernatants of the hybridoma clone (No. 8) and the hybridoma clone (No. 16). These hybridomas are named ESM-8 cells and ESM-16 cells, respectively, and the monoclonal antibodies produced by these hybridomas are respectively ESM-8 antibody (ESM-8 monoclonal antibody) and ESM-16 antibody (ESM-16). Monoclonal antibody).

[Example 2]
Screening for Anti-67LR _161-170 Peptide Antibody-Producing Hybridoma _Having Anticancer Activity Mouse melanoma cell line B16 was seeded in 96 well plate at 1.0 × 10 ⁴ cells / mL, and 24% in 5% FCS-DMEM medium. Pre-cultured for hours. Next, the culture supernatant was removed by aspiration, and 50 μl / well of the culture supernatant of each hybridoma clone and 250 μL / well of 12% FCS-DMEM were added instead. After 96 hours of culture, mitochondrial dehydrogenase activity at 430 nm was measured by the WST-1 method. Hybridoma culture supernatant not producing anti-67LR _161-170 peptide antibody was added to Control. WST-1 was purchased from Roche (Basel, Switzerland) and stored frozen at −20 ° C. under light shielding before use. The microplate reader used was Sic System (Tokyo, Japan).
The results are shown in FIG. FIG. 2 shows the results of measurement of anti-67LR _161-170 peptide antibody in the culture supernatant obtained from each hybridoma clone. As is clear from FIG. 2, strong growth inhibitory activity was observed in the culture supernatant of the hybridoma clone (No. 8).

[Example 3]
To confirm the specificity for _{67LR 161-170} peptide specific ESM-8 antibody to _{67LR 161-170} peptide _{antibodies, 67LR 161-170} peptide (IPCNNKGAHS, SEQ ID NO: 3) and control peptide (MNAANVGWNGSTFA, SEQ ID NO: 4) Binding to was measured using an ELISA method. That is, 67LR _161-170 peptide or control peptide was diluted with 50 μM carbonate buffer so as to be 10 μg / mL, and 100 μL was added to each well of the Immuno plate, followed by incubation at 37 ° C. for 2 hours. Thereafter, the plate was washed with TPBS, 1% BSA-TPBS as a blocking agent was added in an amount of 300 μL to each well, and the plate was left at 4 ° C. overnight. After washing with TPBS, 50 μL / well of 4 μg / mL, 8 μg / mL, and 16 μg / mL ESM-8 antibody was added and reacted at 37 ° C. for 1 hour. After washing with TPBS, 100 μL of HRP-labeled goat anti-mouse IgG (Abcam, Cambridge, UK) (10000-fold dilution) was added and reacted at 37 ° C. for 1 hour. After washing with TPBS, 100 μL of the chromogenic substrate solution was added and reacted at 37 ° C. for 15 minutes, and then the absorbance at 405 nm was measured. The results are shown in FIG. FIG. 3 is a graph showing the results of measuring the binding of the ESM-8 antibody to the 67LR _161-170 peptide and the control peptide by ELISA. As is apparent from FIG. 3, it was found that the ESM-8 antibody did not bind to the control peptide but specifically bound to the 67LR _161-170 peptide.
The ESM-16 antibody was similarly tested. The results are shown in FIG. FIG. 4 is a graph showing the results of measuring the binding of the ESM-16 antibody to the 67LR _161-170 peptide and the control peptide by ELISA. As is apparent from FIG. 4, it was found that the ESM-16 antibody did not bind to the control peptide but specifically bound to the 67LR _161-170 peptide.

[Example 4]
Identification of DNA sequence of heavy and light chain variable regions of monoclonal antibody and preparation of recombinant antibody expression vector mRNA was prepared from ESM-8 cells and ESM-16 cells obtained in Example 1, and cDNA was prepared. The nucleotide sequences of the antibody heavy chain and light chain variable region DNAs were determined using an antibody-specific primer set (Genescript, Piscataway, NJ). The DNA base sequence was determined with a DNA sequencer. The base sequence or amino acid sequence of the heavy chain, light chain variable region and CDR DNA of the ESM-8 antibody are shown in FIG. 5 and Table 1 (SEQ ID NOs: 5 to 20). The base sequence or amino acid sequence of the heavy chain, light chain variable region and CDR DNA of the ESM-16 antibody are shown in FIG. 6 and Table 1 (SEQ ID NOs: 21 to 36). Subsequently, the DNA sequence of the heavy chain variable region of the gene of the ESM-8 antibody or ESM-16 antibody is converted into the DNA of the variable region of the light chain of the mouse IgG ₁ heavy chain constant region and the ESM-8 antibody gene or ESM-16 antibody. Recombinant ESM-8 antibody and recombinant ESM-16 antibody expression vectors were prepared by linking the sequences to the mouse kappa light chain constant region genes and further incorporating them into pCep4 vector (Invitrogen, San Diego, USA).

[Example 5]
Preparation of ESM-8 Antibody and Recombinant ESM-8 Antibody 500 μL of pristane (Funakoshi Co., Ltd.) was intraperitoneally administered to BALB / c mice. Six days later, ESM-8 cells were administered intraperitoneally at 1 × 10 ⁷ cells / mL, and after confirming that the abdomen of the mouse was enlarged, the abdomen was dissected to collect ascites. Ascites was applied to a HiTrap Protein A column (GE Healthcare, UK) to purify the ESM-8 antibody. For purification, the column was equilibrated with a binding buffer (100 mM Sodium Phosphate / 2.5 M NaCl pH 7.4), and ascites diluted 10-fold with the binding buffer was fed to the column. After washing the column with the binding buffer, the ESM-8 antibody was eluted with an elution buffer (100 mM sodium citrate, pH 5.6). The eluted ESM-8 antibody solution was neutralized with a neutralization buffer (1M Tris-HCl, pH 9) (hereinafter, the antibody obtained by this method is simply referred to as ESM-8 antibody, and Differentiated from recombinant ESM-18 antibody). On the other hand, the ESM-8 antibody expression vector obtained in Example 3 was introduced into HEK293 cells (ATCC, Manassas, VA) using Fugene 6 (Promega, Fitchburg, Wisconsin) to produce cells producing recombinant ESM-8 antibody. did. Recombinant ESM-8 antibody was prepared from the culture supernatant of HEK293 cells producing this ESM-8 antibody.

[Example 6]
Measurement of anti-cancer activity The human acute myeloid leukemia cell line HL60 is 10% fetal bovine serum (FCS) (BILOGICAL INDUSTRIES) -added RPMI RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) at 37 ° C. and 5% CO ₂ saturated with water vapor Passaged and maintained under conditions. Cells were maintained in culture in the logarithmic growth phase. RPMI1640 medium used for the culture was 10.4 g of RPMI1640 medium (Cosmo Bio Co., Ltd.), 2.38 g of HEPES (Wako Pure Chemical Industries, Ltd.) per liter of ultrapure water, 200,000 units of penicillin G potassium for injection (Meiji Seika Co., Ltd.) Co., Ltd.) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd., Tokyo) 0.1 vial, NaHCO ₃ (Nacalai Tesque Co., Ltd.) 2.0 g were suspended and then sterilized by filter. Thereafter, fetal calf serum (FCS) was added to RPMI 1640 medium and used for cell culture. HL60 was seed | inoculated to 96 well plate so that it might become 2.0 * 10 < ⁴ > cells / mL (100 microliter / well). Next, Control mIgG (R & D systems, Inc., Minneapolis, USA) or ESM-8 antibody (0.4 μg / mL, 0.8 μg / mL) was added from the top at 100 μL / well. After 96 hours of culture, live cells were detected by ATPlite (PerkinElmer, Inc., MA, USA). In addition, as a control for the 67LR activator, a test section was named in which green tea catechin EGCG was named.
The results are shown in FIG. FIG. 7 is a graph showing the results of examining the cell growth inhibitory activity of ESM-8 antibody against human acute myeloid leukemia cell line HL60. As shown in FIG. 7, it can be seen that in the presence of the ESM-8 antibody, cell proliferation against the human acute myeloid leukemia cell line HL60 is suppressed. Therefore, it can be seen that the ESM-8 antibody suppresses the growth of the human acute myeloid leukemia cell line HL60, like EGCG.

[Example 7]
Evaluation of cancer cell surface binding activity of antibody The binding of ESM-8 antibody to the cell surface was evaluated by flow cytometry analysis. The flow cytometer used was FACSalbibur (Becton, Dickinson and Company, New Jersey, USA). HL60 was prepared to 1.0 × 10 ⁶ cells / mL and then suspended in 1% FCS-PBS containing 0.4 μg / mL of ESM-8 antibody and Control mIgG (R & D systems, Inc., Minneapolis, USA). Incubated for 2 hours on ice. After removing the supernatant by centrifugation (300 × g, 5 min), the suspension was suspended in AF488-labeled rabbit anti-mIgG (x1000) (Invitrogen, San Diego, USA), and incubated on ice for 1 hour. After washing twice with PBS, it was suspended again in PBS and detected by flow cytometry analysis.
In addition, U266 cells were prepared to 1.0 × 10 ⁷ cells / mL, then suspended in RPMI 1640 medium containing 5 μg / mL of ESM-16 antibody and Isotype Control mIg (Cosmo Bio Inc.), and 2 times on ice. Time incubation was performed. After removing the supernatant by centrifugation (300 × g, 5 min), the suspension was suspended in a secondary antibody solution goat anti-mouse IgM Alexa fluor 488 (Invitrogen) and incubated on ice for 1 hour. After washing twice with PBS, it was suspended again in PBS and detected by flow cytometry analysis.
The results for the ESM-8 antibody are shown in FIG. 8, and the results for the ESM-16 antibody are shown in FIG. As shown in FIG. 8, since cells were detected by flow cytometry in the presence of EMS-18 antibody, it was found that EMS-8 antibody binds to the cell surface of human acute myeloid leukemia cell line HL60. It was. Further, as shown in FIG. 9, cells are detected by flow cytometry in the presence of EMS-16 antibody, so that ESM-16 antibody binds to the cell surface of human multiple myeloma cell line U266 cell. I understood.

[Example 8]
Acid Sphingomyelinase Activation Ability of Antibody HL60 was passaged in RPMI RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal calf serum (FCS) under conditions of 5% CO ₂ with water vapor saturation. , Maintained. Cells were maintained in culture in the logarithmic growth phase. RPMI1640 medium used for the culture was 10.4 g of RPMI1640 medium (Cosmo Bio Co., Ltd.), 2.38 g of HEPES (Wako Pure Chemical Industries, Ltd.) per liter of ultrapure water, 200,000 units of penicillin G potassium for injection (Meiji Seika Co., Ltd.) Co., Ltd.) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd.) 0.1 vial, NaHCO ₃ (Nacalai Tesque Co., Ltd.) 2.0 g were suspended and then filter sterilized. Thereafter, fetal calf serum (FCS) was added to RPMI 1640 medium and used for cell culture. BODIPY-C12-Sphingomyelin (1 mg / mL; dissolved in DMSO) was purchased from Sigma (St. Louis, MO). After centrifugal concentration, it was dissolved in methanol to 1 mM and stored at −20 ° C. 20 × 20 silica gel plates were purchased from Merck (Darmstadt, Germany). Cell Lysis Buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% (v / v) Triton-X100, 1 mM EDTA, 50 mM NaF, 30 mM Na ₄ P ₂ O ₇ , 1 mM Phenylmethylsulfuric fluoride, 2 μg / mL Aprotin; The cells were lysed at pH 4.5), stirred by inversion at 4 ° C. for 1 hour, and centrifuged (15000 × g, 30 minutes) to recover the supernatant. For protein quantification, BCA Protein Assay Reagent was used and adjusted to 20 μg / sample. A substrate solution (1 mM BODIPY-C12-Sphingomyelin, 10% Triton X-100, 1M sodium acetate (pH 4.5), dH ₂ O) was added to each sample so that BODIPY-C12-Sphingomyelin was 400 pmol / sample, and the temperature was 37 ° C. The enzyme substrate reaction was carried out for 16 hours. The reaction was stopped by adding 60 μL of CHCl ₃ —CH ₃ OH 2: 1 (v / v), centrifuged (12000 × g, 4 ° C., 5 minutes), then 10 μL of the lower layer was spotted onto a silica gel plate, and the developing solution (CH ₃ Cl—CH ₃ OH—H ₂ O 65: 25: 4 (v / v / v)).
HL60 cells were seeded in 24 well plate at 5 × 10 ⁵ cells / mL, and 1% supplemented with Control mIgG (R & D systems, Inc., Minneapolis, USA) or ESM-8 antibody (final concentration 0.4 μg / ml) Treated with FCS-RPMI1640 medium for 3 hours. Thereafter, the cells were collected and lysed in a cell lysis buffer having a pH of 4.5. The cell lysate was reacted with BODIPY-C12-Sphingomyelin as a substrate for 16 hours, and then the activity of acidic sphingomyelinase was measured by TLC analysis.
The results are shown in FIGS. 10 (a) and 10 (b). As is clear from FIG. 10 (b), it was found that the ESM-8 antibody had about three times the sphingomyelinase activation ability compared to the control.

[Example 9]
Evaluation of Effect of Anti-67LR Antibody on Cancer Cell Growth Inhibitory Effect of ESM-8 Antibody It has been reported that EGCG exhibits anticancer activity via 67LR (Non-patent Documents 18 to 21). Thus, it was examined whether the ESM-8 antibody exerts anticancer activity via 67LR as in EGCG.
The human acute myeloid leukemia cell line HL60 was subcultured in RPMI RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal calf serum (FCS) under conditions of 5% CO ₂ with water vapor saturation. Maintained. Cells were maintained in culture in the logarithmic growth phase. RPMI1640 medium used for the culture was 10.4 g of RPMI1640 medium (Cosmo Bio Co., Ltd.), 2.38 g of HEPES (Wako Pure Chemical Industries, Ltd.) per liter of ultrapure water, 200,000 units of penicillin G potassium for injection (Meiji Seika Co., Ltd.) Co., Ltd.) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd.) 0.1 vial, NaHCO ₃ (Nacalai Tesque Co., Ltd.) 2.0 g were suspended and then filter sterilized. Thereafter, fetal calf serum (FCS) was added to RPMI 1640 medium and used for cell culture.
HL60 was seeded on a 96-well plate at 1.0 × 10 ⁴ cells / mL, and precultured in 5% FCS-RPMI medium for 24 hours. Thereafter, the original medium was aspirated and replaced with 1% FCS-RPMI medium containing Control mIgM (Cosmo Bio Inc.) or anti-67LR monoclonal antibody MLuC5 (Abcam, Cambridge, UK) (30 μg / mL). Let stand for hours. Thereafter, the medium was added at 100 μL / well so as to obtain a 1% FCS-RPMI medium containing Control mIgG (R & D systems, Inc., Minneapolis, USA) or ESM-8 antibody (0.4 μg / mL). After 96 hours, viable cells were detected by ATP lite (PerkinElmer, Inc., MA, USA).
The results are shown in FIG. As can be seen from FIG. 11, when not pretreated with anti-67LR monoclonal antibody, ESM-8 antibody showed cytostatic activity against human acute myeloid leukemia cell line HL60, but pretreated with anti-67LR monoclonal antibody. In that case, ESM-8 cells did not show cytostatic activity on HL60. From these results, it was shown that the EMS-8 antibody exerts anticancer activity through binding to 67LR, and that the ESM-8 antibody functions as a 67LR agonist like EGCG.

[Example 10]
Recombinant ESM-8 antibody inhibits cell proliferation against human acute myeloid leukemia cell line HL60, human pancreatic cancer cell line PANC-1 and human mesothelioma cell line MESO-4 10% human acute myeloid leukemia cell line HL60 It was subcultured and maintained at 37 ° C. under 5% CO ₂ with water vapor saturation in RPMI RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) supplemented with fetal calf serum (FCS) (BIOLOGICAL INDUSTRIES). Cells were maintained in culture in the logarithmic growth phase. RPMI1640 medium used for the culture was 10.4 g of RPMI1640 medium (Cosmo Bio Co., Ltd.) per liter of ultrapure water, 2.38 g of HEPES (Wako Pure Chemical Industries, Ltd.), 200,000 units of penicillin G for injection (Meiji Seika) Co., Ltd.) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd., Tokyo) 0.1 vial, NaHCO ₃ (Nacalai Tesque Co., Ltd.) 2.0 g were suspended and then sterilized by filter. Thereafter, fetal calf serum (FCS) was added to RPMI 1640 medium and used for cell culture. HL60 was seeded on a 96-well plate (100 μL / well) so as to be 1.0 × 10 ⁴ cells / mL, and cultured for 24 hours. Next, Control mIgG (R & D systems, Inc., Minneapolis, USA) or recombinant ESM-8 antibody was added to 1 μg / mL. After 96 hours of culture, live cells were detected by ATPlite (PerkinElmer, Inc., MA, USA).
The human pancreatic cancer cell line PANC-1 was subcultured in RPMI RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal calf serum (FCS) under conditions of 5% CO ₂ with water vapor saturation. , Maintained. Cells were maintained in culture in the logarithmic growth phase. RPMI1640 medium used for the culture was 10.4 g of RPMI1640 medium (Cosmo Bio Co., Ltd.), 2.38 g of HEPES (Wako Pure Chemical Industries, Ltd.) per liter of ultrapure water, 200,000 units of penicillin G potassium for injection (Meiji Seika Co., Ltd.) Co., Ltd.) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd.) 0.1 vial, NaHCO ₃ (Nacalai Tesque Co., Ltd.) 2.0 g were suspended and then filter sterilized. Thereafter, fetal calf serum (FCS) was added to RPMI 1640 medium and used for cell culture. PANC-1 cells were seeded on a 96-well plate (100 μL / well) at 1.0 × 10 ⁴ cells / mL and cultured for 24 hours. Next, Control mIgG (R & D systems, Inc., Minneapolis, USA) or ESM-8 antibody was added to 1 μg / mL. After 96 hours of culture, live cells were detected by ATPlite (PerkinElmer, Inc., MA, USA).
The human mesothelioma cell line MESO-4 was subcultured in RPMI1640 medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal bovine serum (FCS) under a 5% CO ₂ condition saturated with water vapor. Maintained. Cells were maintained in culture in the logarithmic growth phase. RPMI1640 medium used for the culture was 10.4 g of RPMI1640 medium (Cosmo Bio Co., Ltd.), 2.38 g of HEPES (Wako Pure Chemical Industries, Ltd.) per liter of ultrapure water, 200,000 units of penicillin G potassium for injection (Meiji Seika Co., Ltd.) Co., Ltd.) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd.) 0.1 vial, NaHCO ₃ (Nacalai Tesque Co., Ltd.) 2.0 g were suspended and then filter sterilized. Thereafter, fetal calf serum (FCS) was added to RPMI 1640 medium and used for cell culture. MESO-4 cells were seeded on a 96-well plate at 100 × 10 ⁴ cells / mL (100 μL / well) and cultured for 24 hours. Next, Control mIgG (R & D systems, Inc., Minneapolis, USA) or ESM-8 antibody was added to 1.0 μg / mL. After 96 hours of culture, live cells were detected by ATPlite (PerkinElmer, Inc., MA, USA).
The results are shown in FIG. FIG. 12 is a graph showing the results of examining the cell growth inhibitory activity of recombinant ESM-8 antibody on human acute myeloid leukemia cell line HL60, human pancreatic cancer cell line PANC-1 and human mesothelioma cell line MESO-4. It is. As is clear from FIG. 12, the recombinant ESM-8 antibody has cytostatic activity against human acute myeloid leukemia cell line HL60, human pancreatic cancer cell line PANC-1 and human mesothelioma cell line MESO-4. It was found to have

[Example 11]
Acidic sphingomyelinase activation ability of the recombinant ESM-8 antibody The ability to activate acidic sphingomyelinase was evaluated in the same manner as in Example 8 except that the ESM-8 antibody was replaced with the recombinant ESM-8 antibody. The results are shown in FIG. As is clear from FIG. 13, it was found that the recombinant ESM-8 antibody had a higher ability to activate sphingomyelinase than the control.

[Example 12]
Evaluation of the binding activity of the recombinant ESM-8 antibody to the surface of the acute myeloid leukemia cell line HL60 cell The binding of the recombinant ESM-8 antibody to the cell surface was evaluated by flow cytometry analysis. The same flow cytometer as in Example 7 was used. After preparing human acute myeloid leukemia cell line HL60 to 1.0 × 10 ⁶ cells / mL, 1% FCS-PBS to which recombinant ESM-8 antibody or Control mIgG was added to a final concentration of 1 μg / mL was added. Suspended and incubated on ice for 7 h. After removing the supernatant by centrifugation (300 xg, 5 min), the suspension was suspended in AF488-labeled rabbit anti-mIgG (x300) and incubated on ice for 1 h. After washing twice with PBS, it was suspended again in PBS and detected by flow cytometry analysis.
The results are shown in FIG. As shown in FIG. 14, since cells are detected by flow cytometry in the presence of the recombinant ESM-8 antibody, the recombinant ESM-8 antibody can bind to the cell surface of the acute myeloid leukemia cell line HL60 cell. I understood.

[Example 13]
Anti-cancer activity of recombinant ESM-8 antibody in combination with PDE3 inhibitor, PDE5 inhibitor and sphingosine kinase inhibitor PANC-1 cells were added to RPMI RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% FCS. It was subcultured and maintained at 37 ° C. under 5% CO ₂ with water vapor saturation. Cells were maintained in culture in the logarithmic growth phase. RPMI1640 medium used for the culture was 10.4 g of RPMI1640 medium (Cosmo Bio Co., Ltd.), 2.38 g of HEPES (Wako Pure Chemical Industries, Ltd.) per liter of ultrapure water, 200,000 units of penicillin G potassium for injection (Meiji Seika Co., Ltd.) Co., Ltd.) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd.) 0.1 vial, NaHCO ₃ (Nacalai tesque, Inc. Kyoto) 2.0 g was suspended and then filter sterilized. FCS was then added to RPMI 1640 medium and used for cell culture.
PANC-1 cells were seeded on a 96-well plate at 100 × 10 ⁴ cells / mL (100 μL / well). Next, phosphodiesterase 3 (PDE3) inhibitor (Trequinsin (trequinsin), 1.25 μM Calbiochem La Jolla, CA), phosphodiesterase 5 (PDE5) (Vardenafil (Vardenafil), 5 μM TRC Canada), sphingosine kinase inhibitor (Safingin kinase ol (Safine) Goal) Pretreatment was performed with 5 μM Alabaster, AL). After 3 hours, 100 μL / well of Control mIgG (R & D systems, Inc., Minneapolis, USA) or recombinant ESM-8 antibody was added. After 96 hours of culture, live cells were detected by ATPlite (PerkinElmer, Inc., MA, USA).
The results are shown in FIG. As shown in FIG. 15, when a PDE3 inhibitor, a PDE5 inhibitor, and a sphingosine kinase inhibitor are used in combination, the number of living cells is reduced. From this, it was found that a PDE3 inhibitor, a PDE5 inhibitor, and a sphingosine kinase inhibitor enhance the anticancer effect of the recombinant SM-8 antibody.

[Example 14]
Anti-cancer activity of recombinant ESM-8 antibody in combination with gemcitabine Using human mesothelioma cell line MESO-4 instead of human pancreatic cancer cell line PANC-1 cell, using 1 μM gemcitabine as inhibitor, ESM-8 The same procedure as in Example 12 was performed except that the recombinant ESM-8 antibody was used instead of the antibody, and the anticancer activity of the recombinant ESM-8 antibody was evaluated.
The results are shown in FIG. As shown in FIG. 16, the number of viable cells decreased when gemcitabine was used in combination. This indicates that gemcitabine enhances the anticancer effect of recombinant ESM-8 antibody.

[Example 15]
Cancer cell growth inhibitory activity of ESM-16 antibody Human multiple myeloma cells U266 were saturated with water vapor at 37 ° C. in RPMI RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal calf serum (FCS) (BILOGICAL INDUSTRIES). Passaged and maintained under 5% CO ₂ conditions. Cells were maintained in culture in the logarithmic growth phase. RPMI1640 medium used for the culture was 10.4 g of RPMI1640 medium (Cosmo Bio Co., Ltd.), 2.38 g of HEPES (Wako Pure Chemical Industries, Ltd.) per liter of ultrapure water, 200,000 units of penicillin G potassium for injection (Meiji Seika Co., Ltd.) Co., Ltd.) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd.) 0.1 vial, NaHCO ₃ (Nacalai Tesque Co., Ltd.) 2.0 g were suspended and then filter sterilized. Thereafter, fetal calf serum (FCS) was added to RPMI 1640 medium and used for cell culture. U266 cells were seeded on a 96-well plate at 5.0 × 10 ⁴ cells / mL (100 μL / well). Next, Control mIgG (R & D systems, Inc., Minneapolis, USA) or ESM-16 antibody was added to 300 ng / mL. After 96 hours of culture, live cells were detected by ATPlite (PerkinElmer, Inc., MA, USA).
The mouse melanoma cell line B16 was subcultured and maintained in DMEM medium (Kyokuto Pharmaceutical Co., Ltd.) supplemented with 10% fetal calf serum (FCS) at 37 ° C. under 5% CO ₂ with water vapor saturation. Cells were maintained in culture in the logarithmic growth phase. The DMEM medium used for the culture was 3.7 g of NaHCO ₃ (Wako Pure Chemical Industries, Ltd.), 100 U / mL penicillin (Meiji Seika Co., Ltd.), 100 μg / mL streptomycin (Meiji pharmaceutical Company), and 1 L of DMEM medium. 25 mM HEPES (Wako Pure Chemical Industries, Ltd.) was added. Cells were maintained in culture in the logarithmic growth phase. B16 cells were seeded on a 96-well plate at 100 × 10 ⁴ cells / mL (100 μL / well). After 24 hours, Control mIgM (Cosmo Bio Inc.) or ESM-16 antibody was added to 300 ng / mL. After 96 hours of culture, live cells were detected by ATPlite (PerkinElmer, Inc., MA, USA).
The human pancreatic cancer cell line PANC-1 was subcultured in RPMI RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal calf serum (FCS) under conditions of 5% CO ₂ with water vapor saturation. , Maintained. Cells were maintained in culture in the logarithmic growth phase. The RPMI 1640 medium used for the culture was 10.4 g of RPMI 1640 medium (Cosmo Bio Inc.), 2.38 g of HEPES (Wako Pure Chemical Industries, Ltd.) per liter of ultrapure water, 200,000 units of penicillin G potassium for injection (Meiji). Confectionery Co., Ltd.) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd.) 0.1 vial, NaHCO ₃ (Nacalai Tesque Co., Ltd.) 2.0 g were suspended, and then filter sterilized. Thereafter, fetal calf serum (FCS) was added to RPMI 1640 medium and used for cell culture. PANC-1 cells were seeded on a 96-well plate at 100 × 10 ⁴ cells / mL (100 μL / well). Next, Control mIgM (Cosmo Bio, Tokyo, Japan) or ESM-16 antibody was added to PANC-1 cells at 300 ng / mL. After 96 hours of culture, live cells were detected by ATPlite (PerkinElmer, Inc., MA, USA).
The results are shown in FIG. As shown in FIG. 17, in the presence of the EMS-16 antibody, the number of viable cells was any against human multiple myeloma cell U266, mouse melanoma cell line B16, and human pancreatic cancer cell line PANC-1. Was shown to decrease. This indicates that the EMS-16 antibody has cytostatic activity against human multiple myeloma cell U266, mouse melanoma cell line B16 and human pancreatic cancer cell line PANC-1.

Claims (25)

1. An antibody having anticancer activity by activating 67 kDa laminin receptor protein (hereinafter referred to as 67LR).
2. EGCG sensing motif (hereinafter referred to as ESM) -8 An antibody comprising the variable region of an antibody as an antigen-binding region.
3. An antibody comprising the variable region of the ESM-16 antibody as an antigen-binding region.
4. The antibody according to any one of claims 1 to 3, which is a humanized antibody.
5. The antibody according to any one of claims 1 to 3, which is a canine antibody.
6. The antibody according to any one of claims 1 to 3, which is a feline antibody.
7. An antibody comprising the peptide sequence of the complementarity determining region (CDR) of the variable region of the ESM-8 antibody.
8. An antibody comprising a peptide sequence of the complementary determining region (CDR) of the variable region of the ESM-16 antibody.
9. The amino acid sequence of the antibody according to any one of claims 1 to 8, wherein one or more amino acids are substituted, deleted, added and / or inserted, and according to any one of claims 1 to 8. An antibody having an activity equivalent to that of the antibody.
10. A chimeric antigen receptor containing the peptide sequence of the variable region of the ESM-8 antibody.
11. A chimeric antigen receptor comprising the peptide sequence of the variable region of the ESM-16 antibody.
12. The amino acid sequence of the antibody according to any one of claims 1 to 9, wherein one or more amino acids are substituted, deleted, added and / or inserted, and according to any one of claims 1 to 9. A chimeric antigen receptor having an activity equivalent to that of the antibody.
13. The green tea catechin EGCG has any one of the anti-cancer activity, anti-obesity, anti-arteriosclerotic action, anti-allergy, anti-inflammatory or muscle atrophy inhibition physiological activity expressed through 67LR. The antibody according to item.
14. The antibody according to any one of claims 1 to 9, which has an apoptosis-inducing activity by activating acidic sphingomyelinase directly involved in apoptosis induction of green tea catechin EGCG.
15. An anticancer agent comprising a combination of the antibody according to any one of claims 1 to 9 and any one of a PDE3 inhibitor, a PDE5 inhibitor, a Shpk1 inhibitor, or a gemcitabine.
16. 10. A diagnostic antibody for discriminating a person who has applied the antibody according to any one of claims 1 to 9.
17. An antibody obtained by binding a radioisotope, a fluorescent agent, an enzyme, a cytotoxin, or the like to the antibody according to any one of claims 1 to 9.
18. A method for obtaining an antibody using a peptide sequence on the 67LR molecule to which green tea catechin EGCG binds as an immunizing antigen.
19. A method for obtaining an antibody exhibiting anticancer activity using a peptide sequence on the 67LR molecule to which green tea catechin EGCG binds as an immunizing antigen.
20. Anti-cancer activity, anti-obesity, anti-arteriosclerotic action, anti-allergy, anti-inflammatory or muscle atrophy inhibiting physiological activity expressed by green tea catechin EGCG via 67LR with the peptide sequence on 67LR molecule to which green tea catechin EGCG binds A screening method for an antibody having any of the above.
21. Antibody in which any of anti-cancer activity, anti-obesity, anti-arteriosclerotic effect, anti-allergy, anti-inflammatory or muscular atrophy inhibiting physiological activity is enhanced by combining with PDE3 inhibitor, PDE5 inhibitor, Shpk1 inhibitor, or gemcitabine Screening method.
22. An antibody obtained by the method according to any one of claims 18 to 21.
23. A method for treating cancer, obesity, arteriosclerosis, allergy, inflammation or muscle atrophy, comprising administering the antibody according to any one of claims 1 to 9 to a subject.
24. The gene of the ESM-8 antibody is selected from the group consisting of human-derived cell lines, hamster-derived cell lines, mouse-derived cell lines, insect-derived cell lines, sugar chain biosynthesis mutant cell lines of these cell lines, and Escherichia coli and yeast. A method for producing an antibody according to any one of claims 1 to 9, comprising introducing the antibody into a host cell, culturing the host cell to express the antibody, and collecting the expressed antibody.
25. The gene of ESM-16 antibody is selected from the group consisting of human-derived cell lines, hamster-derived cell lines, mouse-derived cell lines, insect-derived cell lines, sugar chain biosynthesis mutant cell lines of these cell lines, and Escherichia coli and yeast. A method for producing an antibody according to any one of claims 1 to 9, comprising introducing the antibody into a host cell, culturing the host cell to express the antibody, and collecting the expressed antibody.

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