Cancer treatment drugs

Abstract

Provided are: a therapeutic drug for hepatocellular carcinoma, a therapeutic method, etc., which can exert a strong and lasting anti-tumor effect compared with existing multi-kinase inhibitors. The present invention is characterized in that it is a combination drug for treating hepatocellular carcinoma, which comprises: lenvatinib or its prodrug, or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof; and an antibody against human DLK-1 having anti-tumor activity in vivo or an antibody fragment derived from the antibody.

Description

Technical Field

[0001] The present invention relates to drugs and methods for treating hepatocellular carcinoma. Background Art

[0002] Liver cancer is the second leading cause of cancer-related deaths worldwide, with approximately 750,000 people dying from it each year. The number of new patients is approximately 780,000 per year, of which approximately 80% are concentrated in Asia, including Japan and China. It is reported that hepatocellular carcinoma accounts for 85-90% of all liver cancers. In Japan, the number of patients with hepatocellular carcinoma is approximately 42,000, and the number of deaths is approximately 26,000 per year. Unresectable hepatocellular carcinoma has limited treatment options and a very poor prognosis, making it a disease with high unmet medical needs.

[0003] As a therapeutic drug for unresectable progressive hepatocellular carcinoma, a multikinase inhibitor targeting multiple receptor tyrosine kinases is generally used. Specifically, sorafenib and lenvatinib are used as first-line drugs, and regorafenib and cabozantinib are used as second-line drugs.

[0004] Lenvatinib is a multikinase inhibitor. It is an orally administered tyrosine kinase inhibitor that selectively inhibits receptor tyrosine kinases related to tumor angiogenesis or tumor malignancy, such as PDGFRα, KIT, and RET, as well as VEGFR1, VEGFR2, and VEGFR3 as vascular endothelial growth factor receptors (VEGFRs), and FGFR1, FGFR2, FGFR3, and FGFR4 as fibroblast growth factor receptors (FGFRs). In March 2018, it was approved in Japan as the world's first primary treatment for unresectable progressive hepatocellular carcinoma. It was approved in the United States and Europe in August 2018. It should be noted that when lenvatinib is used as a drug, it is usually used in the form of mesylate.

[0005] The results of an open-label, randomized, phase III trial (REFLECT trial) comparing lenvatinib with sorafenib as initial treatment for unresectable hepatocellular carcinoma showed that the efficacy of the lenvatinib group was 40.6%, which was higher than the 12.4% of sorafenib. In addition, in the REFLECT trial, the efficacy of the Japanese group (81 people) assigned to the lenvatinib group was 46.9% (see non-patent document 1).

[0006] Thus, lenvatinib showed an efficacy of up to 40.6% for unresectable hepatocellular carcinoma, but no effect was confirmed in the remaining approximately 60% of patients. In addition, the efficacy of sorafenib, another initial treatment drug, was as low as 12.4%, and the therapeutic effect of multikinase inhibitor monotherapy was limited.

[0007] Non-patent literature 1: Kudo M, et al., Lancet, vol.391(10126), p.1163-1173, 2018 Summary of the invention

[0008] Problem that the invention aims to solve

[0009] Under such circumstances, the development of therapeutic drugs, therapeutic methods, and the like for hepatocellular carcinoma that can exert stronger and more prolonged antitumor effects than existing multikinase inhibitors has been desired.

[0010] Solutions for solving problems

[0011] The present invention has been made in view of the above circumstances, and provides the following combination drugs for treating hepatocellular carcinoma, etc.

[0012] (1) A combination drug for treating hepatocellular carcinoma, comprising:

[0013] Lenvatinib or its prodrug, or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, and

[0014] An antibody against human DLK-1 or an antibody fragment derived from the antibody having anti-tumor activity in vivo.

[0015] (2) The combination drug according to (1) above, wherein the tumor is hepatocellular carcinoma.

[0016] (3) The combination drug according to (1) or (2) above, wherein the antibody is a chimeric antibody or a humanized antibody.

[0017] (4) The combination drug according to any one of (1) to (3) above, wherein the antibody is at least one selected from the group consisting of the following antibodies:

[0018] (a) an antibody in which the amino acid sequences of CDR1 to 3 of the H chain V region (heavy chain variable region) are the amino acid sequences shown in SEQ ID NOs: 3 to 5, respectively, and the amino acid sequences of CDR1 to 3 of the L chain V region (light chain variable region) are the amino acid sequences shown in SEQ ID NOs: 6 to 8, respectively,

[0019] (b) an antibody in which the amino acid sequences of CDR1 to 3 of the H chain V region are the amino acid sequences shown in SEQ ID NOs: 9 to 11, respectively, and the amino acid sequences of CDR1 to 3 of the L chain V region are the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively,

[0020] (c) an antibody whose H chain V region amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 16 and whose L chain V region amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 18,

[0021] (d) an antibody whose H chain V region amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 20 and whose L chain V region amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 22,

[0022] (e) an antibody whose H chain V region amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 24 or 26, and whose L chain V region amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 28,

[0023] (f) an antibody whose H chain V region amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 30, 32, 34 or 36, and whose L chain V region amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 46,

[0024] (g) an antibody whose H chain V region amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 38, 40, 42 or 44, and whose L chain V region amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 46,

[0025] (h) an antibody produced by a hybridoma having a deposit number of FERM BP-10899,

[0026] (i) an antibody produced by a hybridoma having a deposit number of FERM BP-10707,

[0027] (j) an antibody produced by a hybridoma having a deposit number of FERM BP-10900, and

[0028] (k) An antibody produced by the hybridoma with the deposit number FERM BP-11337.

[0029] (5) The combination drug according to any one of (1) to (4) above, wherein the antibody or antibody fragment is in the form of a complex with a compound having antitumor activity and / or cytotoxic activity.

[0030] (6) The combination drug according to any one of (1) to (5) above, wherein the combination drug can inhibit cancer cell proliferation or shrink or eliminate tumors even after administration of the combination drug is completed.

[0031] (7) lenvatinib or its prodrug, or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, and

[0032] Antibodies against human DLK-1 or antibody fragments derived therefrom having anti-tumor activity in vivo,

[0033] Use for the manufacture of a medicament for the treatment of hepatocellular carcinoma.

[0034] (8) A method for treating hepatocellular carcinoma, comprising administering to a subject:

[0035] Lenvatinib or its prodrug, or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, and

[0036] An antibody against human DLK-1 or an antibody fragment derived from the antibody having anti-tumor activity in vivo.

[0037] (9) A kit for treating hepatocellular carcinoma, comprising:

[0038] Lenvatinib or its prodrug, or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, and

[0039] An antibody against human DLK-1 or an antibody fragment derived from the antibody having anti-tumor activity in vivo.

[0040] Effects of the Invention

[0041] According to the present invention, a therapeutic agent and a therapeutic method can be provided that can exert a strong and lasting anti-tumor effect on hepatocellular carcinoma compared with existing multi-kinase inhibitors. The therapeutic agent and the therapeutic method of the present invention can exert an effect on patients who could not expect a therapeutic effect in the past, and are extremely useful in this respect. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1A This is a diagram showing the results of a test for measuring the combined effect of lenvatinib and an anti-hDLK-1 antibody (HuBA-1-3D antibody) using a Hep3B xenograft model in this Example.

[0043] Figure 1B The graph shows the results of the increase and decrease in tumor volume of each individual (38 days after transplantation) in the test for measuring the combined effect of lenvatinib and anti-hDLK-1 antibody (HuBA-1-3D antibody) using the Hep3B transplanted tumor model in this Example.

[0044] Figure 1CThe graphs show the weights of tumors excised after the test and photographs of the combined effect measurement test of lenvatinib and anti-hDLK-1 antibody (HuBA-1-3D antibody) using the Hep3B transplant tumor model in this Example.

[0045] Figure 2A This is a diagram showing the results of a test for measuring the combined effect of lenvatinib and an anti-hDLK-1 antibody (HuBA-1-3D antibody) using a HepG2 transplant tumor model in this Example.

[0046] Figure 2B It is a graph showing the tumor volume of the lenvatinib-administered group, the anti-hDLK-1 antibody-administered group, and the lenvatinib and anti-hDLK-1 antibody-administered group on the 34th day after tumor transplantation in the combined effect measurement experiment of lenvatinib and anti-hDLK-1 antibody (HuBA-1-3D antibody) using the HepG2 transplant tumor model in this example.

[0047] Figure 3 The graphs show the results of sensitivity of lenvatinib to Hep3B and HepG2 using the Hep3B transplant tumor model and the HepG2 transplant tumor model. A shows the results using the Hep3B transplant tumor model, and B shows the results using the HepG2 transplant tumor model. DETAILED DESCRIPTION

[0048] Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and can be implemented by appropriately changing on the basis of the following examples without prejudice to the main contents of the present invention. It should be noted that this specification includes the entire contents of the Special Application No. 2019-070120 specification (filed on April 1, 2019) which is the basis for the priority claim of this application. All publications cited in this specification, such as prior art documents and public gazettes, patent gazettes and other patent documents, are incorporated into this specification as a reference.

[0049] 1. Summary of the Invention

[0050] As mentioned above, lenvatinib is an orally administrable multi-kinase inhibitor that has selective inhibitory activity on receptor tyrosine kinases related to tumor angiogenesis or tumor malignancy, such as PDGFRα, KIT, and RET, which are platelet-derived growth factor receptors (PDGFRs), in addition to vascular endothelial growth factor receptors (VEGFR1-3) and fibroblast growth factor receptors (FGFR1-4). In March 2018, it was approved in Japan as the world's first initial treatment for unresectable progressive hepatocellular carcinoma, and in August 2018 it was approved in the United States and Europe. In the REFRECT trial, lenvatinib showed an efficacy of up to 40.6%, but no effect was confirmed in the remaining approximately 60% of patients, and the therapeutic effect of lenvatinib monotherapy was limited.

[0051] In order to develop a therapeutic drug that can exert a strong and lasting anti-tumor effect compared with existing multi-kinase inhibitors, the present inventors conducted experiments and studies using transplanted tumor treatment models using Hep3B cell lines and HepG2 cell lines derived from human hepatocellular carcinoma. As a result, it was clarified that by combining lenvatinib with an antibody against human DLK-1 (delta-like1homolog (Drosophila), δ-like 1 homolog (Drosophila); hereinafter sometimes referred to as "hDLK-1"), a lasting and significantly stronger tumor growth inhibition and tumor reduction effect was exerted compared with the case of administering lenvatinib or the antibody alone.

[0052] Here, hDLK-1 is known to be a single-pass transmembrane type I membrane protein composed of 383 amino acid residues, and is expressed in adult cancers such as hepatocellular carcinoma, small cell lung cancer, pancreatic cancer, and breast cancer, and pediatric tumors such as neuroblastoma, hepatoblastoma, rhabdomyosarcoma, and viral tumors. On the other hand, in normal tissues and organs, the expression of DLK-1 is limited to the adrenal glands, pituitary glands, etc., and is not expressed in most organs, so it is suitable as a therapeutic target molecule for cancer. To date, anti-hDLK-1 monoclonal antibodies that show tumor cell proliferation inhibition and tumor cell death induction effects including excellent tumor shrinkage effects have been developed, and anti-tumor activity has been shown by administering the antibody alone in multiple transplant tumor treatment models using human cancer cell lines.

[0053] According to the combination drug / combination therapy of lenvatinib and anti-hDLK-1 antibody discovered by the present inventors, sufficient therapeutic effects can be expected even for hepatocellular carcinoma patients who could not expect sufficient therapeutic effects (no response) when multikinase inhibitors were administered alone in the past. The present invention was thus completed.

[0054] 2. Combination drugs for cancer treatment

[0055] As described above, the combination drug for treating hepatocellular carcinoma of the present invention (hereinafter sometimes referred to as "the combination drug of the present invention") is characterized by comprising as an active ingredient:

[0056] Lenvatinib or its prodrug, or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof (hereinafter sometimes referred to as "lenvatinib, etc." in this specification) and

[0057] An anti-hDLK-1 antibody or an antibody fragment derived from the antibody having anti-tumor activity in vivo (hereinafter sometimes referred to as "anti-hDLK-1 antibody, etc." in the present specification).

[0058] It should be noted that the present invention also includes: (i) a method for treating hepatocellular carcinoma, which includes the steps of using lenvatinib, etc. and anti-hDLK-1 antibodies, etc., specifically, for example, administering an effective amount of lenvatinib, etc. and anti-hDLK-1 antibodies, etc. to a subject (a hepatocellular carcinoma patient or a patient with a concern (risk of developing the disease) or such a non-human mammal); (ii) the use of lenvatinib, etc. and anti-hDLK-1 antibodies, etc. for the manufacture of a drug for treating hepatocellular carcinoma; (iii) the use of lenvatinib, etc. and anti-hDLK-1 antibodies, etc. for treating hepatocellular carcinoma; and (iv) lenvatinib, etc. and anti-hDLK-1 antibodies, etc. for treating hepatocellular carcinoma.

[0059] In the present invention, the treatment of hepatocellular carcinoma includes, for example, inhibiting the progression of hepatocellular carcinoma, improving the prognosis of hepatocellular carcinoma, and / or preventing the recurrence of hepatocellular carcinoma.

[0060] (1) Lenvatinib, etc.

[0061] As the active ingredient of the combination drug of the present invention, lenvatinib and the like can be known commercial products, but are not limited thereto, and can be synthesized, extracted, purified, etc. by oneself. In the case of self-synthesis, the synthesis method described in the specification of U.S. Patent No. 7,612,092 can also be referred to.

[0062] It should be noted that the official name (IUPAC name) of lenvatinib is 4-[3-chloro-4-(cyclopropylcarbamoylamino)phenoxy]-7-methoxy-quinoline-6-carboxamide, which is represented by the following structural formula.

[0063]

[0064] As the active ingredient of the combination drug of the present invention, a lenvatinib derivative can also be used together with lenvatinib or in place of lenvatinib. As the derivative, any derivative that is considered to be a lenvatinib derivative based on the technical common sense of a person skilled in the art, such as having a chemical structure derived from lenvatinib, is not limited, and preferably has the same degree of anti-tumor activity as lenvatinib. In the present invention, when only lenvatinib is referred to, it means that a derivative of lenvatinib may be included.

[0065] As lenvatinib used in the present invention, in addition to metabolizers such as those that undergo oxidation, reduction, hydrolysis or conjugation in the body, it also includes compounds that undergo oxidation, reduction or hydrolysis in the body to generate lenvatinib (so-called prodrugs). In the present invention, a prodrug refers to a pharmaceutically acceptable compound obtained by modifying a parent compound with a group commonly used in a prodrug, for example, a compound that is endowed with properties such as improved stability and durability and can be expected to be converted into a parent compound in the intestine and show an effect. For example, a prodrug of lenvatinib can be manufactured in the following manner: using a prodrug reagent such as a corresponding halide, using a conventional method to appropriately introduce a group constituting a prodrug into any one or more groups selected from the prodrug-capable groups (e.g., hydroxyl, amino, other groups) in the compound according to a conventional method, and then separating and purifying as needed to manufacture. Here, the group constituting the prodrug is not limited, and preferred examples include lower alkyl-CO-, lower alkyl-O-lower alkylene-CO-, lower alkyl-OCO-lower alkylene-CO-, lower alkyl-OCO-, and lower alkyl-O-lower alkylene-OCO-.

[0066] As the active ingredient of the combination drug of the present invention, these pharmaceutically acceptable salts may be used together with lenvatinib or a prodrug thereof, or may be used instead of lenvatinib or a prodrug thereof.

[0067] The pharmaceutically acceptable salt is not limited, and preferably includes organic sulfonates (e.g., methanesulfonates (also called mesylate), trifluoromethanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates, and camphorsulfonates), organic carboxylates (e.g., acetates, trifluoroacetates, maleates, tartrates, fumarates, and citrates), amino acid salts (e.g., aspartates and glutamates), quaternary ammonium salts, alkali metal salts (e.g., sodium salts and potassium salts), inorganic acid salts (e.g., sulfates, nitrates, perchlorates, phosphates, carbonates, and bicarbonates), hydrohalides (e.g., hydrochlorides, hydrobromides, and hydroiodides), alkaline earth metal salts (e.g., magnesium salts and calcium salts), etc. As a preferred embodiment of the pharmaceutically acceptable salt of lenvatinib used in the present invention, lenvatinib mesylate can be included.

[0068] The lenvatinib used in the present invention also includes all isomers that can be obtained in the structure of the compound (for example, geometric isomers, optical isomers based on asymmetric carbon, rotational isomers, stereoisomers, and tautomers, etc.) and mixtures of two or more of these isomers, and is not limited to the description of the structural formula for convenience. In addition, lenvatinib can be any of the S-body, R-body or RS-body, without limitation. Furthermore, lenvatinib sometimes exists in the form of hydrates and solvates depending on its type. In the present invention, the hydrates and solvates are also included in lenvatinib and can be used as the active ingredient of the combination drug of the present invention. As the solvate, there is no limitation, for example, solvates with ethanol, etc. can be listed.

[0069] In the combination drug of the present invention, the content ratio of lenvatinib and the like as the active ingredient is not limited and can be appropriately set, for example, it can be set in the range of 0.01 to 99 weight %, preferably 0.01 to 30 weight %, more preferably 0.05 to 20 weight %, and further preferably 0.1 to 10 weight % relative to the entire combination drug. By setting the content ratio of the active ingredient within the above range, the combination drug of the present invention can fully exert the therapeutic effect of hepatocellular carcinoma.

[0070] The combination drug of the present invention may contain any other multikinase inhibitors and the like in addition to lenvatinib and the like, within a range that does not significantly impair the effects of the present invention.

[0071] (2) Anti-hDLK-1 antibodies, etc.

[0072] The anti-hDLK-1 antibody (antibody against human DLK-1 having anti-tumor activity in vivo) which is the active ingredient of the combination drug of the present invention can be prepared based on the following description.

[0073] (i) Preparation of Antigen

[0074] The amino acid sequence of hDLK-1 (SEQ ID NO: 2) is published on the NCBI (GenBank) website (http: / / www.ncbi.nlm.nih.gov / ) as "Accession No.: NP_003827". In addition, the base sequence encoding the amino acid sequence of hDLK-1 (SEQ ID NO: 1) is published on the same website as "Accession No.: NM_003836".

[0075] As an antigen, a polypeptide or peptide (also referred to as a peptide) containing at least a portion (all or a part) of the amino acid sequence of hDLK-1 can be used, and preferably a peptide containing at least a portion (all or a part) of the amino acid sequence of the extracellular region (FA-1) of hDLK-1 can be used. As described above, the extracellular region of hDLK-1 is a region containing six EGF-like motifs (EGF-1 to EGF-6), and is a region containing amino acids at positions 24 to 244 in the amino acid sequence shown in SEQ ID NO: 2, preferably a region consisting of amino acids at positions "24" to "248 to 285" in the amino acid sequence shown in SEQ ID NO: 2 (approximately 225 to 262 amino acid residues).

[0076] Here, for the peptide used as an antigen, the length of the above-mentioned "at least a part of the amino acid sequence" is not particularly limited, and for example, it is preferably a region containing one or more of the six EGF-like motifs. More preferred are, for example, a region containing EGF-1 and EGF-2 (i.e., a region consisting of amino acids at positions 24 to 91 in the amino acid sequence shown in SEQ ID NO: 2), a region containing EGF-3 and EGF-4 (i.e., a region consisting of amino acids at positions 92 to 167 in the amino acid sequence shown in SEQ ID NO: 2), and a region containing EGF-4, EGF-5, and EGF-6 (i.e., a region consisting of amino acids at positions 131 to 244 in the amino acid sequence shown in SEQ ID NO: 2).

[0077] The peptide used as an antigen may be prepared by chemical synthesis or by genetic engineering using Escherichia coli or the like, and any method known to those skilled in the art may be used.

[0078] When the peptide is chemically synthesized, it can be synthesized by a known method for peptide synthesis. In addition, the synthesis can also be performed using any of solid phase synthesis and liquid phase synthesis. A commercially available peptide synthesis device (e.g., PSSM-8, manufactured by Shimadzu Corporation, etc.) can also be used.

[0079] When synthesizing a peptide by genetic engineering, first, a DNA encoding the peptide is designed and synthesized. The design and synthesis can be performed, for example, as follows: using a vector containing a full-length hDLK-1 gene as a template, using primers designed to synthesize a desired DNA region, by PCR. Then, a recombinant vector for protein expression is obtained by connecting the above DNA to an appropriate vector, and the recombinant vector is introduced into a host to allow the target gene to be expressed, thereby obtaining a transformant (Molecular cloning 4th Ed. Cold Spring Harbor Laboratory Press (2012)).

[0080] As a vector, a phage or plasmid that can self-propagate in a host microorganism is used. In addition, animal virus and insect virus vectors can be used. The preparation of a recombinant vector can be carried out as follows, i.e., the purified DNA is cut with an appropriate restriction enzyme, and the restriction enzyme site of an appropriate vector DNA is inserted and connected to the vector. As a host used during transformation, there is no particular limitation as long as it is a host that can express the target gene. For example, bacteria (Escherichia coli, Bacillus subtilis, etc.), yeast, animal cells (COS cells, CHO cells, etc.), insect cells or insects can be listed. Mammals such as goats can also be used as hosts. Methods for introducing recombinant vectors into hosts are known.

[0081] Then, the transformant is cultured, and the peptide used as an antigen is collected from the culture. "Culture" refers to any of culture supernatant, cultured cells or cultured bacterial bodies, or their fragments.

[0082] After the culture, when the target peptide is produced in the bacteria or cells, the peptide is extracted by disrupting the bacteria or cells. In addition, when the target peptide is produced outside the bacteria or cells, the bacteria or cells are removed directly using the culture solution or by centrifugation. Then, the target peptide can be isolated and purified by using a general biochemical method that can be used for the separation and purification of peptides, such as ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, etc., alone or in combination.

[0083] In the present invention, a cell-free protein synthesis system can also be used to obtain a peptide as an antigen by in vitro translation. In this case, two methods can be used: a method using RNA as a template and a method using DNA as a template (transcription / translation). As a cell-free protein synthesis system, a commercially available system can be used, such as the ExpresswayTM system (Invitrogen), PURESYSTEM (registered trademark; Post Genome Research Institute), and the TNT system (registered trademark; Promega Corporation).

[0084] The peptide obtained as described above can also be bound to an appropriate carrier protein, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), human thyroglobulin, chicken γ-globulin, and the like.

[0085] In addition, the antigen may be a peptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of hDLK-1 (SEQ ID NO: 2) or a partial sequence of the aforementioned sequence. For example, a peptide consisting of an amino acid sequence in which one or more (preferably one or several (for example, one to ten, more preferably one to five)) amino acids are deleted, one or more (preferably one or several (for example, one to ten, more preferably one to five)) amino acids are replaced by other amino acids, or one or more (preferably one or several (for example, one to ten, more preferably one to five)) other amino acids are added in the amino acid sequence of hDLK-1 or a partial sequence thereof may also be used.

[0086] In the present invention, genes to be introduced into cells etc. include genes encoding hDLK-1 protein or partial fragments thereof, or mutant proteins or fragments thereof. As such a gene, for example, a gene having the base sequence shown in SEQ ID NO: 1 or a partial sequence thereof can be used.

[0087] Furthermore, as a gene to be introduced into cells or the like, a nucleotide sequence or a partial sequence thereof which hybridizes under stringent conditions with a sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having hDLK-1 activity can also be used.

[0088] "Stringent conditions" refer to the conditions for washing after hybridization, and refer to the conditions where the salt (sodium) concentration of the buffer is 10 to 500 mM and the temperature is 42°C to 72°C, preferably the conditions where the salt concentration is 50 to 300 mM and the temperature is 55 to 68°C.

[0089] In order to introduce a mutation into a gene, it can be carried out by a known method such as the Kunkel method or the gapped duplex method, and using, for example, a mutation introduction kit utilizing a site-directed mutagenesis method, such as GeneTailorTM Site-Directed Mutagenesis System (manufactured by Invitrogen), TaKaRa Site-Directed Mutagenesis System (Prime STAR (registered trademark) Mutagenesis Basal kit, Mutan (registered trademark) -Super Express Km, etc.: manufactured by TAKARA BIO).

[0090] (ii) Preparation of polyclonal antibodies

[0091] The prepared antigen is administered to a mammal for immunization. The mammal is not particularly limited, and examples thereof include rats, mice, and rabbits, among which mice are preferred.

[0092] The amount of antigen administered to each animal can be appropriately set according to the presence or absence of an adjuvant. As adjuvants, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminum hydroxide adjuvant, etc. can be listed. Immunization can be mainly performed by injection into the vein, sole, subcutaneous, intraperitoneal cavity, etc. Moreover, the interval between immunizations is not particularly limited, and 1 to 10 immunizations, preferably 2 to 3 immunizations, are performed at intervals of several days to several weeks, preferably at intervals of 1 week. Then, 3 to 7 days after the last immunization date, the antibody titer is measured by enzyme immunoassay (ELISA or EIA) or radioimmunoassay (RIA), and blood can be collected on the day when the desired antibody titer appears to obtain antiserum. In the above-mentioned antibody extraction method, when it is necessary to purify the antibody, it can be purified by appropriately selecting known methods such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration chromatography, affinity chromatography, or by combining these. Then, the reactivity of the polyclonal antibody in the antiserum is measured by ELISA method, etc.

[0093] (iii) Preparation of monoclonal antibodies

[0094] Collection of antibody-producing cells

[0095] The anti-hDLK-1 antibody of the present invention is not limited, but is preferably a monoclonal antibody.

[0096] The prepared antigen is administered to mammals for immunization, such as rats, mice, and rabbits. The amount of antigen administered to each animal can be appropriately set depending on the presence or absence of an adjuvant. The adjuvant is the same as described above. The immunization method is also the same as described above. Then, 1 to 60 days after the last immunization day, preferably 1 to 14 days after, antibody-producing cells are extracted. Antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, among which lymph node cells and spleen cells are preferred.

[0097] Cell fusion

[0098] In order to obtain hybridoma (antibody-producing cell strain), cell fusion of antibody-producing cells and myeloma cells is performed. As the myeloma cells fused with antibody-producing cells, generally available established cell strains of animals such as mice can be used. As the cell strain used, it is preferred to have drug selectivity, a cell strain that cannot survive in HAT selection medium (containing hypoxanthine, aminopterin and thymidine) under an unfused state and can only survive under a state fused with antibody-producing cells.

[0099] Examples of myeloma cells include mouse myeloma cell lines such as P3-X63-Ag8.653, P3-X63-Ag8 (X63), P3-X63-Ag8.U1 (P3U1), P3 / NS I / 1-Ag4-1 (NS1), and Sp2 / 0-Ag14 (Sp2 / 0). Myeloma cells can be selected by appropriately considering compatibility with antibody-producing cells.

[0100] Then, myeloma cells and antibody-producing cells were subjected to cell fusion. For cell fusion, 1×10 6 ~1×10 7 Antibody-producing cells / mL and 2×10 5 ~2×10 6 / mL of myeloma cells. The cell ratio of antibody-producing cells to myeloma cells (antibody-producing cells: myeloma cells) is not limited, but is generally preferably 1:1 to 10:1, more preferably 3:1. Then, a fusion reaction is carried out in the presence of a cell fusion promoter. As a cell fusion promoter, for example, polyethylene glycol with an average molecular weight of 1000 to 6000 Daltons (D) can be used. In addition, a commercially available cell fusion device using electrical stimulation (such as electroporation) can also be used to fuse antibody-producing cells with myeloma cells.

[0101] Hybridoma selection and cloning

[0102] The target hybridoma is selected from the cells after the cell fusion treatment. As a method, the cell suspension is appropriately diluted with RPMI-1640 medium containing fetal bovine serum, etc., and then inoculated on a microtiter plate, and a selection medium is added to each well, and the selection medium is appropriately replaced thereafter for cultivation. As a result, cells that begin to grow around 14 days after the start of cultivation in the selection medium can be used as hybridomas.

[0103] Then, the culture supernatant of the hybridoma that has been gradually proliferated is screened for the presence of antibodies that react with hDLK-1. The screening of hybridomas can be carried out according to conventional methods, without particular limitation. For example, a portion of the culture supernatant contained in the well where the hybridoma has grown can be collected and screened by ELISA, EIA, RIA, etc.

[0104] The fused cells can be cloned by limiting dilution method, etc. Antibodies showing strong reactivity with hDLK-1 are determined by flow cytometry, etc., and hybridomas producing the antibodies are selected to establish clones.

[0105] Collection of monoclonal antibodies

[0106] As a method for culturing established hybridomas and extracting monoclonal antibodies from the obtained cultures, conventional cell culture methods or ascites formation methods can be adopted. "Cultivation" means growing hybridomas in a culture dish or culture bottle, or proliferating hybridomas in the abdominal cavity of animals as follows.

[0107] In the cell culture method, hybridomas can be cultured in an animal cell culture medium such as RPMI-1640 medium, MEM medium or serum-free medium containing 10% fetal bovine serum under normal culture conditions (e.g., 37°C, 5% CO2 concentration) for 7 to 14 days, and antibodies can be obtained from the culture supernatant.

[0108] In the ascites formation method, approximately 1×10 7 Furthermore, it is preferred to extract ascites after 2 to 3 weeks.

[0109] In the above-mentioned antibody extraction method, when it is necessary to purify the antibody, it can be purified by appropriately selecting a known method such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration, affinity chromatography, or a combination thereof.

[0110] Selection of clones with anti-tumor activity

[0111] The anti-hDLK-1 antibody used in the present invention is an antibody having anti-tumor activity in vivo.

[0112] Here, "anti-tumor activity" refers to the activity of killing tumor cells (cancer cells) or the activity of inhibiting tumor growth. In the present invention, as the anti-tumor activity, for example, the tumor angiogenesis inhibition activity can be preferably mentioned. As the types of human tumors (tumor cells) in which the anti-hDLK-1 antibody of the present invention can exert anti-tumor activity, there can be listed: known human tumors in which the expression of hDLK-1 has been confirmed.

[0113] The confirmation of antitumor activity in vivo can be carried out, for example, as follows: using tumor-bearing mice in which the desired tumor cells are transplanted subcutaneously, the antibodies obtained as described above are administered to the mice. In this case, the administration of the antibody can be carried out immediately after the transplantation of the tumor cells (prevention model), or after confirming that the transplanted tumor has reached a predetermined volume (treatment model). There is no restriction on the method of administration, for example, intraperitoneal administration at 20 mg / kg body weight can be performed once every three days. In the case of the prevention model, the presence and level of antitumor activity can be evaluated by the frequency of tumor formation and tumor volume. In the case of the treatment model, the presence and level of antitumor activity can be evaluated based on tumor volume and tumor weight.

[0114] In the present invention, the anti-hDLK-1 antibody having anti-tumor activity in vivo is not particularly limited, and for example, anti-hDLK-1 antibodies disclosed in WO2008 / 056833, WO2009 / 116670, and WO2014 / 054820 can be used.

[0115] More specifically, for example, the following anti-hDLK-1 antibodies (a) to (k) are mentioned, and at least one of these can be used.

[0116] (a) An antibody wherein the amino acid sequences of CDR1 to 3 of the H chain V region are the amino acid sequences shown in SEQ ID NOs: 3 to 5, respectively, and the amino acid sequences of CDR1 to 3 of the L chain V region are the amino acid sequences shown in SEQ ID NOs: 6 to 8, respectively.

[0117] (b) An antibody in which the amino acid sequences of CDR1 to 3 of the H chain V region are the amino acid sequences shown in SEQ ID NOs: 9 to 11, respectively, and the amino acid sequences of CDR1 to 3 of the L chain V region are the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively.

[0118] (c) An antibody wherein the amino acid sequence of the H chain V region consists of the amino acid sequence shown in SEQ ID NO: 16 (the corresponding base sequence is SEQ ID NO: 15), and the amino acid sequence of the L chain V region consists of the amino acid sequence shown in SEQ ID NO: 18 (the corresponding base sequence is SEQ ID NO: 17). It should be noted that the sequences of the CDRs in the H chain V region and the L chain V region of this antibody are the same as those of the antibody described in (a) above.

[0119] (d) An antibody wherein the amino acid sequence of the H chain V region consists of the amino acid sequence shown in SEQ ID NO: 20 (the corresponding base sequence is SEQ ID NO: 19), and the amino acid sequence of the L chain V region consists of the amino acid sequence shown in SEQ ID NO: 22 (the corresponding base sequence is SEQ ID NO: 21). It should be noted that the sequences of each CDR in the H chain V region and the L chain V region of this antibody are the same as those of the antibody described in (b) above.

[0120] (e) An antibody (humanized antibody) wherein the amino acid sequence of the H chain V region consists of the amino acid sequence shown in SEQ ID NO: 24 or 26 (the corresponding base sequence is SEQ ID NO: 23 or 25), and the amino acid sequence of the L chain V region consists of the amino acid sequence shown in SEQ ID NO: 28 (the corresponding base sequence is SEQ ID NO: 27). It should be noted that the sequences of the CDRs in the H chain V region and the L chain V region of this antibody are the same as those of the antibody described in (a) above.

[0121] (f) An antibody (humanized antibody) wherein the amino acid sequence of the H chain V region consists of the amino acid sequence shown in SEQ ID NO: 30, 32, 34 or 36 (the corresponding base sequence is SEQ ID NO: 29, 31, 33 or 35), and the amino acid sequence of the L chain V region consists of the amino acid sequence shown in SEQ ID NO: 46 (the corresponding base sequence is SEQ ID NO: 45). It should be noted that the sequences of each CDR in the H chain V region and the L chain V region of this antibody are the same as those of the antibody described in (b) above.

[0122] (g) An antibody (humanized antibody) wherein the amino acid sequence of the H chain V region consists of the amino acid sequence shown in SEQ ID NO: 38, 40, 42 or 44 (the corresponding base sequence is SEQ ID NO: 37, 39, 41 or 43), and the amino acid sequence of the L chain V region consists of the amino acid sequence shown in SEQ ID NO: 46 (the corresponding base sequence is SEQ ID NO: 45). It should be noted that the sequences of each CDR in the H chain V region and the L chain V region of this antibody are the same as those of the antibody described in (b) above.

[0123] (h) Antibodies produced by the hybridoma with the deposit number FERM BP-10899.

[0124] (i) Antibodies produced by the hybridoma with the accession number FERM BP-10707.

[0125] (j) Antibodies produced by the hybridoma with the deposit number FERM BP-10900.

[0126] (k) An antibody produced by the hybridoma with the deposit number FERM BP-11337.

[0127] Here, regarding the antibody of (f) above, the amino acid sequence represented by SEQ ID NO: 32 is an amino acid sequence in which alanine (A) at position 24 of the amino acid sequence represented by SEQ ID NO: 30 is substituted with glycine (G),

[0128] The amino acid sequence represented by SEQ ID NO: 34 is an amino acid sequence in which threonine (T) at position 74 of the amino acid sequence represented by SEQ ID NO: 30 is substituted with lysine (K).

[0129] The amino acid sequence represented by SEQ ID NO: 36 is an amino acid sequence in which alanine (A) at position 24 of the amino acid sequence represented by SEQ ID NO: 30 is substituted with glycine (G), and threonine (T) at position 74 is substituted with lysine (K).

[0130] Similarly, regarding the antibody of (g) above, the amino acid sequence represented by SEQ ID NO: 40 is an amino acid sequence in which alanine (A) at position 24 of the amino acid sequence represented by SEQ ID NO: 38 is substituted with glycine (G).

[0131] The amino acid sequence represented by SEQ ID NO: 42 is an amino acid sequence in which threonine (T) at position 74 of the amino acid sequence represented by SEQ ID NO: 38 is substituted with lysine (K).

[0132] The amino acid sequence represented by SEQ ID NO: 44 is an amino acid sequence in which alanine (A) at position 24 of the amino acid sequence represented by SEQ ID NO: 38 is substituted with glycine (G), and threonine (T) at position 74 is substituted with lysine (K).

[0133] Such modified antibodies with amino acid substitutions in the antibodies (humanized antibodies) of (f) and (g) above are antibodies with higher affinity (Avidity; antigen binding activity), for example, they can maintain binding activity with cancer cells with low antigen expression on the cell surface. In addition, the modified antibodies can maintain stable antigen binding activity for a long time in liquid formulations and in the blood (plasma) of monkeys and humans.

[0134] In addition, regarding the antibodies (h) to (k) above, the hybridoma with the deposit number FERM BP-11337 is named “mouse-mouse hybridoma BA-1-3D” and was deposited on February 1, 2011, the hybridoma with the deposit number FERM BP-10707 is named “mouse-mouse hybridoma: M3-1” and was deposited on October 18, 2006, the hybridoma with the deposit number FERM BP-10899 is named “mouse-mouse hybridoma DI-2-14” and was deposited on August 21, 2007, and the hybridoma with the deposit number FERM BP-10900 is named “mouse-mouse hybridoma DI-6” and was deposited on August 21, 2007, all of which are deposited in the National Institute of Advanced Industrial Science and Technology (National Institute of Advanced Industrial Science and Technology, International Patent Organism Depository (IPOD, NITE)). Technology) (name of the international depositary authority when the certificate is issued).

[0135] Preferred examples of anti-hDLK-1 antibodies that can be used in the present invention include antibodies that bind to sites (e.g., epitopes) to which the above-mentioned various anti-hDLK-1 antibodies bind (recognize) and antibodies that can compete with the above-mentioned various anti-hDLK-1 antibodies.

[0136] Epitopes of anti-hDLK-1 antibodies

[0137] The epitope (antigenic determinant) of the anti-hDLK-1 antibody is not limited as long as it is at least a part of the antigen hDLK-1. For example, it is preferably at least a part of the region consisting of amino acids 24 to 91 (the region containing EGF-1 to EGF-2 of hDLK-1), the region consisting of amino acids 92 to 167 (the region containing EGF-3 to EGF-4 of hDLK-1), or the region consisting of amino acids 131 to 244 (the region containing EGF-4 to EGF-6 of hDLK-1) in the amino acid sequence of hDLK-1 shown in SEQ ID NO: 2. Among them, the region containing EGF-1 to EGF-2 of hDLK-1 is more preferred. The anti-hDLK-1 antibody that recognizes (binds to) this region has a high internalization activity into tumor cells, for example, and is extremely useful in the use of immunoconjugates described later.

[0138] (iv) Recombinant antibodies and antibody fragments

[0139] Genetically recombinant antibodies

[0140] One preferred embodiment of the anti-hDLK-1 antibody is a genetically recombinant antibody. The genetically recombinant antibody is not limited, but examples thereof include chimeric antibodies and humanized antibodies.

[0141] Chimeric antibodies (i.e., humanized chimeric antibodies) are antibodies in which the variable region of a mouse antibody is connected (joined) to the constant region of human origin (see Proc. Natl. Acad. Sci. USA 81, 6851-6855, (1984) etc.). When making chimeras, such connected antibodies can be easily constructed by genetic recombination technology.

[0142] When making humanized antibodies, the complementarity determining regions (CDRs) are transplanted from the variable regions of mouse antibodies to the human variable regions, and a reconstructed variable region is made in which the framework region (FR) is of human origin and the CDR is of mouse origin (i.e., CDR grafting). Then, these humanized reconstructed human variable regions are connected to the human constant regions. The method for making humanized antibodies can be referred to, for example, Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989); Japanese Patent Publication No. 4-502408 (Japanese Patent Publication No. 2828340; Queen et al.), etc. Furthermore, the present invention also includes amino acids (preferably 1 to several, more preferably 1 to 2 amino acids) in a part of the V region (excluding the CDR sequence) of the H chain or L chain of the above-mentioned humanized antibody replaced with modified amino acids of other amino groups. As a modified humanized anti-hDLK-1 antibody, a humanized antibody with higher affinity (Avidity; antigen binding activity) can also be prepared, for example, it can maintain binding activity with cancer cells with low antigen expression on the cell surface. In addition, the modified humanized anti-hDLK-1 antibody can maintain stable antigen binding activity for a long time in a liquid formulation and in the blood (plasma) of monkeys and humans.

[0143] Among the chimeric antibodies and humanized antibodies described above, it is preferred that, for example, the N-glycosidic bond complex sugar chain in the antibody Fc region is a sugar chain to which fucose is not bound to the N-acetylglucosamine at the reducing end of the sugar chain. Specifically, an antibody comprising the following gene recombinant antibody molecule can be cited, wherein the gene recombinant antibody molecule has a sugar chain in which the 1st position of the fucose is not α-bonded to the 6th position of the N-acetylglucosamine at the reducing end of the N-glycosidic bond complex sugar chain in the Fc region of the antibody molecule. If it is such an antibody, the ADCC activity can be greatly improved. Moreover, this point (the characteristic of the N-glycosidic bond complex sugar chain in the antibody Fc region) is also preferred for the aforementioned polyclonal antibodies and monoclonal antibodies.

[0144] Antibody fragments

[0145] In the present invention, fragments of anti-hDLK-1 antibodies can also be used together with the anti-hDLK-1 antibodies of the present invention or in place of the anti-hDLK-1 antibodies of the present invention. Here, the antibody fragment is preferably one that has binding activity to hDLK-1, similarly to the anti-hDLK-1 antibodies of the present invention (including humanized antibodies other than mouse antibodies), and preferably one that has anti-tumor activity in vivo and recognizes the same epitope as the anti-hDLK-1 antibodies of the present invention, but is not particularly limited thereto.

[0146] The antibody fragment refers to a region of a part of an anti-hDLK-1 polyclonal antibody or an anti-hDLK-1 monoclonal antibody (i.e., an antibody fragment derived from the anti-hDLK-1 antibody of the present invention), and examples thereof include Fab, Fab', F(ab')2, Fv (antibody variable fragment), single-chain antibody (H chain, L chain, H chain V region and L chain V region, etc.), scFv, diabody (scFv dimer), dsFv (disulfide bond stabilized V region), and a peptide containing at least a part of a complementarity determining region (CDR).

[0147] Fab is an antibody fragment having antigen-binding activity with a molecular weight of about 50,000, in which about half of the N-terminal side of the H chain and the entire L chain are bound by a disulfide bond among fragments obtained by treating an antibody molecule with papain, a protease. Fab can also be produced by inserting a DNA encoding Fab of an antibody into an expression vector for prokaryotes or eukaryotes, introducing the vector into prokaryotes or eukaryotes, and expressing the vector.

[0148] F(ab')2 is a fragment obtained by treating an antibody molecule with pepsin, a proteolytic enzyme, and is a slightly larger antibody fragment with a molecular weight of about 100,000 and antigen-binding activity, in which Fab is linked via a disulfide bond in the hinge region. It can also be prepared by forming a thioether bond or a disulfide bond in Fab as described below.

[0149] Fab' is an antibody fragment having antigen-binding activity and a molecular weight of about 50,000, which is obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2. Fab' can also be produced by inserting a DNA encoding the Fab' fragment of an antibody into an expression vector for prokaryotes or eukaryotes, introducing the vector into prokaryotes or eukaryotes, and expressing the vector, thereby producing Fab'.

[0150] scFv is a VH-P-VL or VL-P-VH polypeptide in which one H chain V region (VH) and one L chain V region (VL) are linked by an appropriate peptide linker (P), and is an antibody fragment having antigen binding activity. scFv can be prepared by obtaining cDNA encoding VH and VL of an antibody, constructing a DNA encoding scFv, inserting the DNA into an expression vector for prokaryotes or eukaryotes, and introducing the expression vector into prokaryotes or eukaryotes to express it, thereby producing it.

[0151] Diabodies are antibody fragments formed by dimerization of scFv, which are antibody fragments with bivalent antigen binding activity. The bivalent antigen binding activity can be the same or one of them can be different. Diabodies can be prepared as follows: cDNAs encoding VH and VL of antibodies are obtained, DNA encoding scFv is constructed and the length of the amino acid sequence of P is less than 8 residues, the DNA is inserted into an expression vector for prokaryotes or eukaryotes, and the expression vector is introduced into prokaryotes or eukaryotes to express it, thereby manufacturing.

[0152] dsFv refers to an antibody formed by replacing one amino acid residue in each of VH and VL with a polypeptide having a cysteine ​​residue through a disulfide bond between the cysteine ​​residues. The amino acid residue replaced with a cysteine ​​residue can be selected based on the prediction of the three-dimensional structure of the antibody according to the method disclosed by Reiter et al. (Protein Engineering, 7, 697-704, 1994). dsFv can be prepared as follows: cDNA encoding VH and VL of the antibody is obtained, DNA encoding dsFv is constructed, the DNA is inserted into an expression vector for prokaryotes or an expression vector for eukaryotes, and the expression vector is introduced into a prokaryote or a eukaryote to express it, thereby producing it.

[0153] The peptide containing CDR is composed of at least one region in the CDR (CDR1 to 3) of VH or VL. The peptide containing multiple CDRs can be directly bound or bound via an appropriate peptide linker. The peptide containing CDR can be prepared as follows: construct a DNA encoding the CDR of VH and VL of the antibody, insert the DNA into an expression vector for prokaryotes or eukaryotes, and then introduce the expression vector into a prokaryote or eukaryote to express it, thereby producing it. In addition, the peptide containing CDR can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (tert-butyloxycarbonyl method).

[0154] The antibody fragment used in the present invention may be a part or all of the antibody Fc region containing an N-glycosidic bond complex sugar chain as a sugar chain in which N-acetylglucosamine at the reducing end of the sugar chain is not bound to fucose in its original form, or may be a fusion protein of the above antibody fragment and a part or all of the antibody Fc region containing an N-glycosidic bond complex sugar chain as a sugar chain in which N-acetylglucosamine at the reducing end of the sugar chain is not bound to fucose. Such an antibody fragment is preferred because it can greatly enhance ADCC activity.

[0155] Specific examples of the antibody fragment used in the present invention are not limited, and examples thereof include fragments containing CDR1 to 3 of the H chain V region and CDR1 to 3 of the L chain V region, and fragments containing the entire H chain V region and the entire L chain V region of the various anti-hDLK-1 antibodies described above.

[0156] (v) Antibody-drug complexes

[0157] The anti-hDLK-1 antibody and antibody fragment used in the present invention may be in the form of a complex with a compound having anti-tumor activity and / or cell-killing activity. It should be noted that after the antibody molecule, antibody fragment molecule and the compound having anti-tumor activity and / or cell-killing activity are prepared separately in advance, the substance obtained by compounding them is generally referred to as an immunoconjugate. In addition, by using genetic recombination technology, a protein toxin as a compound having anti-tumor activity and / or cell-killing activity is genetically linked to the gene of an antibody or antibody fragment, and the substance obtained by expressing it as one protein (fusion protein) is generally referred to as an immunotoxin.

[0158] Examples of compounds having antitumor activity include doxorubicin, calicheamicin, mitomycin C, and auristatin E. Examples of compounds having cytotoxic activity include saponin, ricin, Pseudomycin exotoxin, and diphtheria toxin. Among them, saponin and Pseudomycin exotoxin are preferably used.

[0159] The method for preparing the complex is not limited, and examples thereof include a method of coupling an antibody and a drug via a disulfide bond or a hydrazone bond.

[0160] The anti-hDLK-1 antibody used in the present invention has excellent internalization activity into target tumor cells expressing hDLK-1. Therefore, by pre-combining compounds having anti-tumor activity and / or cell-killing activity, these compounds can be directly and highly selectively acted on tumor cells. The complex is extremely excellent in the ability to deliver the drug to the target tumor cells.

[0161] The internalization activity into cells can be evaluated by fluorescently labeling the antibody with rhodamine or the like and observing the intracellular entry behavior and antibody localization using a fluorescence microscope or the like.

[0162] (3) Combination drugs

[0163] The combined drug of the present invention comprises lenvatinib and the like and anti-hDLK-1 antibody and the like as active ingredients.

[0164] The combination drug of the present invention can be administered to humans or non-human mammals (e.g., mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) as test subjects by various administration routes, specifically oral or parenteral (e.g., intravenous injection (intravenous injection), intramuscular injection, intraperitoneal injection, subcutaneous injection, rectal administration, transdermal administration).

[0165] Therefore, the combination drug of the present invention can be used alone, but can be formulated into a suitable dosage form by a conventional method and using a pharmaceutically acceptable carrier according to the administration route.

[0166] As dosage forms, oral preparations include, for example, tablets, powders, fine granules, granules, coated tablets, capsules, oral solutions, suspensions, emulsions, syrups, and lozenges, and parenteral preparations include, for example, injections (including drips), inhalants, ointments, nasal drops, and liposomes.

[0167] As carriers that can be used in the formulation of these preparations, for example, in addition to commonly used excipients, binders, disintegrants, lubricants, colorants and flavoring agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, bulking agents, wetting agents, surfactants, dispersants, buffers, preservatives, solubilizers and analgesics as needed can be listed. Known components that can be used as raw materials for pharmaceutical preparations can be mixed and formulated by conventional methods.

[0168] Examples of nontoxic substances that can be used as the component include: animal and vegetable oils such as soybean oil, beef tallow, and synthetic glycerides; hydrocarbons such as liquid paraffin, squalane, and paraffin wax; ester oils such as octyldodecyl myristate and isopropyl myristate; higher alcohols such as cetostearyl alcohol and behenyl alcohol; silicone resins; silicone oils; polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, and polyoxyethylene-polyoxypropylene Surfactants such as block copolymers; water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinyl pyrrolidone, and methyl cellulose; lower alcohols such as ethanol and isopropanol; polyols such as glycerol, propylene glycol, dipropylene glycol, sorbitol, and polyethylene glycol; sugars such as glucose and sucrose; inorganic powders such as silicic anhydride, magnesium aluminum silicate, and aluminum silicate; inorganic salts such as sodium chloride and sodium phosphate; purified water, etc., can all be salts or hydrates thereof.

[0169] Preferred examples of excipients include lactose, fructose, corn starch, saccharide, glucose, mannitol, sorbitol, crystalline cellulose, and silicon dioxide. Preferred examples of binders include polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, polypropylene glycol / polyoxyethylene block polymers, and meglumine. Preferred examples of disintegrants include Such as starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, and carboxymethylcellulose calcium. As lubricants, preferably, for example, magnesium stearate, talc, polyethylene glycol, silicon dioxide, and hydrogenated vegetable oil are listed. As colorants, preferably, those allowed to be added to drugs are listed. As flavor correctors, preferably, for example, cocoa powder, menthol, incense powder, peppermint oil, borneol and cinnamon powder are listed, all of which can be salts or hydrates thereof.

[0170] The dosage of the combination drug of the present invention can usually be appropriately set to a wide range based on the mixing ratio of the active ingredients in the preparation, taking into account the age, weight, type / progress of the disease, route of administration, number of administrations ( / 1 day), administration period, etc. of the subject (patient) of administration. In addition, the combination drug of the present invention can substantially administer lenvatinib, etc. as an active ingredient, and anti-hDLK-1 antibodies, etc. at the same time, or one of them can be administered first and the other can be administered later, without limitation. In addition, these administrations can be administered in a single dose in the amount contained in the combination drug, or they can be administered continuously, without limitation.

[0171] The case where the combination drug of the present invention is used as a parenteral preparation or an oral preparation will be described below.

[0172] When used as a parenteral agent, the form is generally not limited. In the case of various injections, for example, it can be provided in the form of a unit dose ampoule or a multi-dose container, or in the form of a lyophilized powder that is dissolved again in a dissolving solution when used. In addition to lenvatinib and anti-hDLK-1 antibodies that are active ingredients, the parenteral agent can contain various known excipients and additives in various forms within the range that does not impair the effects of the above-mentioned active ingredients. For example, in the case of various injections, aliphatic polyols such as water, glycerol, propylene glycol, and polyethylene glycol can be listed.

[0173] The dosage (per day) of the parenteral preparation is not limited. For example, in the case of various injections, the active ingredients such as lenvatinib and anti-hDLK-1 antibodies can usually be set to an amount that can be taken by the application object (subject, patient, etc.) per 1 kg body weight of 0.01 to 1000 mg, 0.05 to 500 mg or 0.1 to 50 mg, or can be set to an amount that can be taken by 0.5 to 20 mg or an amount that can be taken by 1 to 10 mg.

[0174] When used in the form of an oral dosage form, the form is usually not limited, and it can be any of the above-mentioned dosage forms, or it can be made into a dry product that is redissolved when used. In the oral dosage form, in addition to lenvatinib and anti-hDLK-1 antibodies that become active ingredients, various known excipients and additives can be contained in various forms within the range that does not damage the effects of the above-mentioned active ingredients. For example, it can be listed as: binders (syrup, gum arabic, gelatin, sorbitol, tragacanth gum, polyvinyl pyrrolidone, etc.), fillers (lactose, sugar, corn starch, potato starch, calcium phosphate, sorbitol, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silicon dioxide, etc.), disintegrants (various starches, etc.), and wetting agents (sodium lauryl sulfate, etc.).

[0175] Regarding the dosage (per day) of the oral preparation, generally, the amount of lenvatinib, etc. and anti-hDLK-1 antibody, etc., which are active ingredients, can be set to an amount that can be taken by the application object (subject, patient, etc.) per 1 kg body weight of 0.05 to 5000 mg, 0.1 to 1000 mg, or 0.1 to 100 mg, or it can be set to an amount that can be taken by 0.5 to 50 mg, or an amount that can be taken by 1 to 10 mg. In addition, the compounding ratio of the active ingredient in the oral preparation is not limited, and can be appropriately set in consideration of the number of administrations per day, etc.

[0176] The combination drug of the present invention can exert a strong and lasting anti-tumor effect in the treatment of hepatocellular carcinoma compared with existing multikinase inhibitors. For example, after the administration of the drug is completed, the proliferation of cancer cells can be inhibited, or the tumor can be reduced or eliminated. The combination drug of the present invention is extremely useful in that it can also exert an effect on hepatocellular carcinoma patients who could not expect a therapeutic effect in the past.

[0177] 3. Kit

[0178] The present invention may also provide a kit for treating hepatocellular carcinoma comprising lenvatinib and the like and an anti-hDLK-1 antibody and the like as components.

[0179] In consideration of stability (storability) and ease of use, lenvatinib and the like and anti-hDLK-1 antibodies and the like in the kit may be provided in a dissolved state, for example.

[0180] In addition to lenvatinib and anti-hDLK-1 antibodies, the kit may contain other components. For example, it may contain a marker substance for an antibody, or a solid-phase reagent fixed with an antibody or its marker, etc. The marker substance for an antibody refers to a substance labeled with an enzyme, a radioisotope, a fluorescent compound, a chemiluminescent compound, etc. In addition, it may also contain various buffers, sterile water, various cell culture containers, various reaction containers (Eppendorf tubes, etc.), blocking agents (serum components such as bovine serum albumin (BSA), skim milk, goat serum, etc.), cleaning agents, surfactants, various plates, preservatives such as sodium azide, and experimental operation manuals (instructions), etc.

[0181] The kit only needs to include at least the aforementioned lenvatinib, etc. and anti-hDLK-1 antibody, etc. As components, the components necessary for treating hepatocellular carcinoma may all be included in the kit together or separately, without limitation.

[0182] Hereinafter, the present invention will be described in more detail by giving examples, but the present invention is not limited thereto.

[0183] Example 1

[0184] Using the Hep3B xenograft tumor model, the combined effect of lenvatinib mesylate and anti-hDLK-1 antibody in tumorigenesis inhibitory activity was investigated.

[0185] Here, lenvatinib mesylate was commercially available (hereinafter referred to as "lenvatinib" in this example). The anti-hDLK-1 antibody used in this example is the "HuBA-1-3D-1-A24G / T73K antibody" described in the WO2014 / 054820 publication (hereinafter referred to as "HuBA-1-3D antibody" in this example). The construction of the DNA encoding the antibody protein is based on the method described in the same publication (Examples in the specification), and then, for the production / preparation of the antibody protein using host cells, GlymaxX (registered trademark) technology (refer to ProBioGen AG; https: / / www.probiogen.de / genetic-glyco-engineering-adcc-glymaxx.html) is used. It should be noted that the HuBA-1-3D-1-A24G / T73K antibody used in this example is an antibody in which the H chain V region is composed of the amino acids shown in sequence number 36 and the L chain V region is composed of the amino acids shown in sequence number 46.

[0186] 1×10 6Hep3B cells were transplanted subcutaneously into the right flank of 6-7 week-old female NOD / ShiJic-scidJcl mice (day 0). When the average tumor volume reached 100 mm 3 At the stage of about 14 days, the patients were divided into the following groups: control group (N=8, 103.5±18.1 mm 3 ), lenvatinib (3 mg / kg body weight) group (N=8, 102.8±16.8 mm 3 ), HuBA-1-3D antibody (1 mg / kg body weight) administration group (N=8, 102.7±15.0 mm 3 ), lenvatinib (3 mg / kg body weight) and HuBA-1-3D antibody (1 mg / kg body weight) combined administration group (N=8, 102.5±12.5 mm 3 )(hereinafter, lenvatinib + HuBA-1-3D antibody administration group), from that day for 12 days, HuBA-1-3D antibody was administered twice a week (a total of 4 times; 14th, 18th, 22nd, 25th day), and lenvatinib was administered 5 times a week (administered for 5 days, rested for 2 days) (a total of 10 times; 14th, 15th, 16th, 17th, 18th, 21st, 22nd, 23rd, 24th, 25th day). Tumor volume was measured twice a week and reached 1500mm 3 The individual stops observing.

[0187] As a result, the tumor volume of the control group was 557.5±240.5 mm on the 25th day after cancer cell transplantation (the day of administration completion, day 25). 3 , while the lenvatinib group was 268.1±175.7mm 3 , HuBA-1-3D antibody administration group: 144.6±111.2mm 3 , lenvatinib + HuBA-1-3D antibody administration group was 66.2±59.1mm 3 Compared with the control group, all the drug groups showed stronger anti-tumor activity. In addition, the efficacy after drug administration was studied. The results showed that on the 31st day after cancer cell transplantation (6 days after drug administration and 31 days), the control group was 1282.8±448.3mm 3 , lenvatinib group: 572.2±456.0mm 3 , HuBA-1-3D antibody administration group: 92.9±79.3mm 3 , while the lenvatinib + HuBA-1-3D antibody group was 40.3±46.1mm 3 Furthermore, on the 38th day after cancer cell transplantation (13 days after the end of administration, 38 days), the lenvatinib-administered group was 1148.5±591.0 mm 3, HuBA-1-3D antibody administration group: 402.3±364.4mm 3 , while the group receiving lenvatinib + HuBA-1-3D antibody was 52.8±63.9mm 3 That is, it was confirmed that compared with the lenvatinib-administered group and the HuBA-1-3D antibody-administered group, the lenvatinib + HuBA-1-3D antibody-administered group continued to have a stronger anti-tumor activity after the end of the administration ( Figure 1A ).

[0188] The changes in tumor volume of each individual are shown in Figure 1B During the dosing period, the increase in tumor volume in the lenvatinib-administered group and the HuBA-1-3D antibody-administered group was suppressed compared with the control group, but after the end of the dosing period, an increase in tumor volume was observed in almost all individuals in the lenvatinib-administered group and the HuBA-1-3D antibody-administered group. In contrast, in the lenvatinib + HuBA-1-3D antibody-administered group, the increase in tumor volume was suppressed in almost all individuals even after the end of the dosing period.

[0189] The weight and photos of the tumors collected on the day of observation cessation (day 38) are shown in Figure 1C . Regarding tumor weight, the lenvatinib-administered group (N=7) was 841.1±515.8 mg, the HuBA-1-3D antibody-administered group (N=8) was 392.3±412.9 mg, and the lenvatinib+HuBA-1-3D antibody-administered group (N=8) was 22.9±40.5 mg. Compared with the lenvatinib-administered group and the HuBA-1-3D antibody-administered group, the tumor weight of the lenvatinib+HuBA-1-3D antibody-administered group was significantly smaller. In addition, compared with the lenvatinib-administered group and the HuBA-1-3D antibody-administered group, the tumor in the lenvatinib+HuBA-1-3D antibody-administered group was smaller, and the disappearance of the tumor was confirmed in half of the individuals.

[0190] Example 2

[0191] Using the HepG2 xenograft tumor model, the combined effect of lenvatinib mesylate and anti-hDLK-1 antibody in tumorigenesis inhibitory activity was investigated.

[0192] Here, lenvatinib mesylate was commercially available (hereinafter referred to as "lenvatinib" in this example). The anti-hDLK-1 antibody used in this example is the "HuBA-1-3D-1-A24G / T73K antibody" described in the WO2014 / 054820 publication (hereinafter referred to as "HuBA-1-3D antibody" in this example). The construction of the DNA encoding the antibody protein is based on the method described in the same publication (Examples in the specification), and then, for the production / preparation of the antibody protein using host cells, GlymaxX (registered trademark) technology is used (refer to ProBioGen AG; https: / / www.probiogen.de / genetic-glyco-engineering-adcc-glymaxx.html). It should be noted that the HuBA-1-3D-1-A24G / T73K antibody used in this example is an antibody in which the H chain V region is composed of the amino acids shown in sequence number 36 and the L chain V region is composed of the amino acids shown in sequence number 46.

[0193] 5×10 6 HepG2 cells were transplanted subcutaneously into the right flank of 7-week-old female NOD / ShiJic-scidJcl mice (day 0). When the average tumor volume reached 100 mm 3 At the stage of about 10 days, the patients were divided into the following groups: control group (N=8, 118.4±15.6 mm 3 ), lenvatinib (10 mg / kg body weight) group (N=8, 118.8±15.2 mm 3 ), HuBA-1-3D antibody (1 mg / kg body weight) administration group (N=8, 119.8±12.9 mm 3 ), lenvatinib (10 mg / kg body weight) and HuBA-1-3D antibody (1 mg / kg body weight) combined administration group (N=8, 119.7±13.4 mm 3 )(hereinafter, lenvatinib + HuBA-1-3D antibody administration group), from that day for 12 days, HuBA-1-3D antibody was administered twice a week (a total of 4 times; days 10, 13, 17, 21), and lenvatinib was administered 5 times a week (administered for 5 days, rested for 2 days) (a total of 10 times; days 10, 11, 12, 13, 14, 17, 18, 19, 20, 21). Tumor volume was measured twice a week and reached 1500mm 3 The individual stops observing.

[0194] As a result, the tumor volume of the control group was 596.1±201.6 mm on the 21st day after cancer cell transplantation (the day of administration completion, day 21).3 (N=8), while the lenvatinib (10 mg / kg body weight) group was 326.2±188.7 mm 3 (N=8), HuBA-1-3D (1 mg / kg) administration group: 116.4±42.4 mm 3 (N=8), lenvatinib (10 mg / kg body weight) + HuBA-1-3D (1 mg / kg) antibody administration group: 110.6±33.3 mm 3 (N=8), compared with the control group, all the drug groups showed stronger anti-tumor activity. Furthermore, the efficacy after drug administration was studied, and the results showed that on the 31st day after cancer cell transplantation (10 days after drug administration and 31 days after administration), the control group was 1427.7±591.7mm 3 (N=8), lenvatinib (10 mg / kg body weight) group: 817.4±583.4 mm 3 (N=8), HuBA-1-3D (1 mg / kg) antibody administration group: 394.0±169.8 mm 3 (N=8), while the group treated with lenvatinib (10 mg / kg body weight) + HuBA-1-3D antibody (1 mg / kg) was 240.8±90.7 mm 3 (N=8). Compared with the HuBA-1-3D (1 mg / kg) administration group and the lenvatinib (10 mg / kg body weight) administration group, the tumor volume of the lenvatinib (10 mg / kg body weight) and HuBA-1-3D (1 mg / kg) combination administration group was significantly smaller, confirming the enhancement of the anti-tumor effect brought about by the combination administration ( Figure 2A At the 31st day time point, 8 subjects in the control group had tumor volumes exceeding 1500 mm. 3 Four individuals in the lenvatinib group were sacrificed, and the tumor volume exceeded 1500mm in the 8 individuals in the lenvatinib group 3 One individual was killed. The combined effect of lenvatinib (10 mg / kg body weight) + HuBA-1-3D (1 mg / kg) was observed at the 31st day, and the lenvatinib (10 mg / kg body weight) and HuBA-1-3D (1 mg / kg) groups were observed until the 34th day (13 days after the end of the administration). The results showed that the tumor volume of the lenvatinib (10 mg / kg body weight) group (N=7) was 796.2±461.7mm 3 , HuBA-1-3D (1 mg / kg) group (N=8) 584.1±241.9 mm 3 , while the lenvatinib (10 mg / kg body weight) + HuBA-1-3D (1 mg / kg) administration group (N=8) was 358.0±168.1 mm 3Compared with the HuBA-1-3D (1 mg / kg) administration group and the lenvatinib (10 mg / kg body weight) administration group, the tumor volume of the lenvatinib (10 mg / kg body weight) and HuBA-1-3D (1 mg / kg) combination administration group was significantly smaller, confirming that the anti-tumor effect was enhanced by combined administration ( Figure 2B ).

[0195] [Reference Example 1]

[0196] The sensitivity of two cell lines (Hep3B and HepG2) derived from human hepatocellular carcinoma to lenvatinib mesylate (commercially available product; hereinafter referred to as "lenvatinib" in this example) was compared using a transplanted tumor treatment model.

[0197] In the Hep3B xenograft model, cells were transplanted subcutaneously into NOD / SCID mice, and the average tumor volume exceeded 100 mm 3 At the time point (14th day), the mice were divided into groups of 8 and the drug was administered from that day. For drug administration, 10 μL / g per unit body weight of the mouse was orally administered using a gastric tube, 5 times a week (5 days of administration and 2 days of rest) (a total of 12 times; 14th, 15th, 16th, 17th, 18th, 21st, 22nd, 23rd, 24th, 25th, 28th, 29th day) until the last day of the experiment. Regarding the tumor volume (mean ± standard deviation) on the 29th day after transplantation (29th day; the last day of the experiment), the medium (water for injection) administration group was 1050.3±553.8mm 3 (N=8), lenvatinib (3 mg / kg) group: 400.9±160.9 mm 3 (N=8), lenvatinib (10 mg / kg) group: 200.2±133.3 mm 3 (N=8), lenvatinib (30 mg / kg) group: 136.2±33.8 mm 3 (N=8). Taking the vehicle group as the benchmark, the tumor volume ratios (T / C) of the lenvatinib-treated groups (3, 10, and 30 mg / kg-treated groups) on day 29 were 38.2% (P<0.05), 19.1% (P<0.05), and 13.0% (P<0.05), respectively. Figure 3 A).

[0198] On the other hand, the HepG2 xenograft model was confirmed to be less sensitive to lenvatinib than the Hep3B model. 3At the time point (10th day), the mice were divided into groups of 8 and the drug was administered from that day. For drug administration, 10 μL / g per unit body weight of the mouse was orally administered using a gastric tube, 5 times a week (5 days of administration and 2 days of rest) (a total of 14 times; 10th, 11th, 12th, 13th, 14th, 17th, 18th, 19th, 20th, 21st, 24th, 25th, 26th, 27th day) until the day before the last day of the experiment. Regarding the tumor volume (mean ± standard deviation) on the 28th day after transplantation (28th day; the last day of the experiment), the medium (water for injection) administration group was 1163.0±205.1mm 3 (N=8), lenvatinib (3 mg / kg) group: 892.5±220.7 mm 3 (N=8), lenvatinib (10 mg / kg) group: 506.8±215.7 mm 3 (N=8), lenvatinib (30 mg / kg) group: 380.0±146.8 mm 3 (N=8). Taking the vehicle group as the benchmark, the tumor volume ratios (T / C) of the lenvatinib-treated groups (3, 10, and 30 mg / kg-treated groups) on day 28 were 76.7% (P<0.05), 43.6% (P<0.05), and 32.7% (P<0.05), respectively. Figure 3 B).

[0199] Industrial Applicability

[0200] The therapeutic agent and method of the present invention can exert a stronger and longer-lasting antitumor effect in the treatment of hepatocellular carcinoma than existing multikinase inhibitors, and are extremely useful in that they can exert an effect on patients for whom no therapeutic effect could be expected in the past.

[0201] SEQ ID NO: 23: Recombinant DNA

[0202] SEQ ID NO: 24: Synthetic construct (recombinant protein)

[0203] SEQ ID NO: 25: Recombinant DNA

[0204] SEQ ID NO: 26: Synthetic construct (recombinant protein)

[0205] SEQ ID NO: 27: Recombinant DNA

[0206] SEQ ID NO: 28: Synthetic construct (recombinant protein)

[0207] SEQ ID NO: 29: Recombinant DNA

[0208] SEQ ID NO: 30: Synthetic construct (recombinant protein)

[0209] SEQ ID NO: 31: Recombinant DNA

[0210] SEQ ID NO: 32: Synthetic construct (recombinant protein)

[0211] SEQ ID NO: 33: Recombinant DNA

[0212] SEQ ID NO: 34: Synthetic construct (recombinant protein)

[0213] SEQ ID NO: 35: Recombinant DNA

[0214] SEQ ID NO: 36: Synthetic construct (recombinant protein)

[0215] SEQ ID NO: 37: Recombinant DNA

[0216] SEQ ID NO: 38: Synthetic construct (recombinant protein)

[0217] SEQ ID NO: 39: Recombinant DNA

[0218] SEQ ID NO: 40: Synthetic construct (recombinant protein)

[0219] SEQ ID NO: 41: Recombinant DNA

[0220] SEQ ID NO: 42: Synthetic construct (recombinant protein)

[0221] SEQ ID NO: 43: Recombinant DNA

[0222] SEQ ID NO: 44: Synthetic construct (recombinant protein)

[0223] SEQ ID NO: 45: Recombinant DNA

[0224] SEQ ID NO: 46: Synthetic construct (recombinant protein)

[0225]

[0226] Sequence Listing <110> Chiome Bioscience Inc. <120> Cancer treatment drugs <130> G2308WO <150> JP 2019-070120 <151> 2019-04-01 <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 1532 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (154)..(1305) <400> 1 gagagcgcag cgcgcagccc ggtgcagccc tggctttccc ctcgctgcgc gcccgcgccc 60 cctttcgcgt ccgcaaccag aagcccagtg cggcgccagg agccggaccc gcgcccgcac 120 cgctcccggg accgcgaccc cggccgccca gag atg acc gcg acc gaa gcc ctc 174 Met Thr Ala Thr Glu Ala Leu 1 5 ctg cgc gtc ctc ttg ctc ctg ctg gct ttc ggc cac agc acc tat ggg 222 Leu Arg Val Leu Leu Leu Leu Leu Ala Phe Gly His Ser Thr Tyr Gly 10 15 20 gct gaa tgc ttc ccg gcc tgc aac ccc caa aat gga ttc tgc gag gat 270 Ala Glu Cys Phe Pro Ala Cys Asn Pro Gln Asn Gly Phe Cys Glu Asp 25 30 35 gac aat gtt tgc agg tgc cag cct ggc tgg cag ggt ccc ctt tgt gac 318 Asp Asn Val Cys Arg Cys Gln Pro Gly Trp Gln Gly Pro Leu Cys Asp 40 45 50 55 cag tgc gtg acc tct ccc ggc tgc ctt cac gga ctc tgt gga gaa ccc 366 Gln Cys Val Thr Ser Pro Gly Cys Leu His Gly Leu Cys Gly Glu Pro 60 65 70 ggg cag tgc att tgc acc gac ggc tgg gac ggg gag ctc tgt gat aga 414 Gly Gln Cys Ile Cys Thr Asp Gly Trp Asp Gly Glu Leu Cys Asp Arg 75 80 85 gat gtt cgg gcc tgc tcc tcg gcc ccc tgt gcc aac aac ggg acc tgc 462 Asp Val Arg Ala Cys Ser Ser Ala Pro Cys Ala Asn Asn Gly Thr Cys 90 95 100 gtg agc ctg gac gat ggc ctc tat gaa tgc tcc tgt gcc ccc ggg tac 510 Val Ser Leu Asp Asp Gly Leu Tyr Glu Cys Ser Cys Ala Pro Gly Tyr 105 110 115 tcg gga aag gac tgc cag aaa aag gac ggg ccc tgt gtg atc aac ggc 558 Ser Gly Lys Asp Cys Gln Lys Lys Asp Gly Pro Cys Val Ile Asn Gly 120 125 130 135 tcc ccc tgc cag cac gga ggc acc tgc gtg gat gat gag ggc cgg gcc 606 Ser Pro Cys Gln His Gly Gly Thr Cys Val Asp Asp Glu Gly Arg Ala 140 145 150 tcc cat gcc tcc tgc ctg tgc ccc cct ggc ttc tca ggc aat ttc tgc 654 Ser His Ala Ser Cys Leu Cys Pro Pro Gly Phe Ser Gly Asn Phe Cys 155 160 165 gag atc gtg gcc aac agc tgc acc ccc aac cca tgc gag aac gac ggc 702 Glu Ile Val Ala Asn Ser Cys Thr Pro Asn Pro Cys Glu Asn Asp Gly 170 175 180 gtc tgc act gac att ggg ggc gac ttc cgc tgc cgg tgc cca gcc ggc 750 Val Cys Thr Asp Ile Gly Gly Asp Phe Arg Cys Arg Cys Pro Ala Gly 185 190 195 ttc atc gac aag acc tgc agc cgc ccg gtg acc aac tgc gcc agc agc 798 Phe Ile Asp Lys Thr Cys Ser Arg Pro Val Thr Asn Cys Ala Ser Ser 200 205 210 215 ccg tgc cag aac ggg ggc acc tgc ctg cag cac acc cag gtg agc tac 846 Pro Cys Gln Asn Gly Gly Thr Cys Leu Gln His Thr Gln Val Ser Tyr 220 225 230 gag tgt ctg tgc aag ccc gag ttc aca ggt ctc acc tgt gtc aag aag 894 Glu Cys Leu Cys Lys Pro Glu Phe Thr Gly Leu Thr Cys Val Lys Lys 235 240 245 cgc gcg ctg agc ccc cag cag gtc acc cgt ctg ccc agc ggc tat ggg 942 Arg Ala Leu Ser Pro Gln Gln Val Thr Arg Leu Pro Ser Gly Tyr Gly 250 255 260 ctg gcc tac cgc ctg acc cct ggg gtg cac gag ctg ccg gtg cag cag 990 Leu Ala Tyr Arg Leu Thr Pro Gly Val His Glu Leu Pro Val Gln Gln 265 270 275 ccg gag cac cgc atc ctg aag gtg tcc atg aaa gag ctc aac aag aaa 1038 Pro Glu His Arg Ile Leu Lys Val Ser Met Lys Glu Leu Asn Lys Lys 280 285 290 295 acc cct ctc ctc acc gag ggc cag gcc atc tgc ttc acc atc ctg ggc 1086 Thr Pro Leu Leu Thr Glu Gly Gln Ala Ile Cys Phe Thr Ile Leu Gly 300 305 310 gtg ctc acc agc ctg gtg gtg ctg ggc act gtg ggt atc gtc ttc ctc 1134 Val Leu Thr Ser Leu Val Val Leu Gly Thr Val Gly Ile Val Phe Leu 315 320 325 aac aag tgc gag acc tgg gtg tcc aac ctg cgc tac aac cac atg ctg 1182 Asn Lys Cys Glu Thr Trp Val Ser Asn Leu Arg Tyr Asn His Met Leu 330 335 340 cgg aag aag aag aac ctg ctg ctt cag tac aac agc ggg gag gac ctg 1230 Arg Lys Lys Lys Asn Leu Leu Leu Gln Tyr Asn Ser Gly Glu Asp Leu 345 350 355 gcc gtc aac atc atc ttc ccc gag aag atc gac atg acc acc ttc agc 1278 Ala Val Asn Ile Ile Phe Pro Glu Lys Ile Asp Met Thr Thr Phe Ser 360 365 370 375 aag gag gcc ggc gac gag gag atc taa gcagcgttcc cacagccccc 1325 Lys Glu Ala Gly Asp Glu Glu Ile 380 tctagattct tggagttccg cagagcttac tatacgcggt ctgtcctaat ctttgtggtg 1385 ttcgctatct cttgtgtcaa atctggtgaa cgctacgctt acatatattg tctttgtgct 1445 gctgtgtgac aaacgcaatg caaaaacaat cctctttctc tctcttaatg catgatacag 1505 aataataata agaatttcat ctttaaa 1532 <210> 2 <211> 383 <212> PRT <213> Homo sapiens <400> 2 Met Thr Ala Thr Glu Ala Leu Leu Arg Val Leu Leu Leu Leu Leu Ala 1 5 10 15 Phe Gly His Ser Thr Tyr Gly Ala Glu Cys Phe Pro Ala Cys Asn Pro 20 25 30 Gln Asn Gly Phe Cys Glu Asp Asp Asn Val Cys Arg Cys Gln Pro Gly 35 40 45 Trp Gln Gly Pro Leu Cys Asp Gln Cys Val Thr Ser Pro Gly Cys Leu 50 55 60 His Gly Leu Cys Gly Glu Pro Gly Gln Cys Ile Cys Thr Asp Gly Trp 65 70 75 80 Asp Gly Glu Leu Cys Asp Arg Asp Val Arg Ala Cys Ser Ser Ala Pro 85 90 95 Cys Ala Asn Asn Gly Thr Cys Val Ser Leu Asp Asp Gly Leu Tyr Glu 100 105 110 Cys Ser Cys Ala Pro Gly Tyr Ser Gly Lys Asp Cys Gln Lys Lys Asp 115 120 125 Gly Pro Cys Val Ile Asn Gly Ser Pro Cys Gln His Gly Gly Thr Cys 130 135 140 Val Asp Asp Glu Gly Arg Ala Ser His Ala Ser Cys Leu Cys Pro Pro 145 150 155 160 Gly Phe Ser Gly Asn Phe Cys Glu Ile Val Ala Asn Ser Cys Thr Pro 165 170 175 Asn Pro Cys Glu Asn Asp Gly Val Cys Thr Asp Ile Gly Gly Asp Phe 180 185 190 Arg Cys Arg Cys Pro Ala Gly Phe Ile Asp Lys Thr Cys Ser Arg Pro 195 200 205 Val Thr Asn Cys Ala Ser Ser Pro Cys Gln Asn Gly Gly Thr Cys Leu 210 215 220 Gln His Thr Gln Val Ser Tyr Glu Cys Leu Cys Lys Pro Glu Phe Thr 225 230 235 240 Gly Leu Thr Cys Val Lys Lys Arg Ala Leu Ser Pro Gln Gln Val Thr 245 250 255 Arg Leu Pro Ser Gly Tyr Gly Leu Ala Tyr Arg Leu Thr Pro Gly Val 260 265 270 His Glu Leu Pro Val Gln Gln Pro Glu His Arg Ile Leu Lys Val Ser 275 280 285 Met Lys Glu Leu Asn Lys Lys Thr Pro Leu Leu Thr Glu Gly Gln Ala 290 295 300 Ile Cys Phe Thr Ile Leu Gly Val Leu Thr Ser Leu Val Val Leu Gly 305 310 315 320 Thr Val Gly Ile Val Phe Leu Asn Lys Cys Glu Thr Trp Val Ser Asn 325 330 335 Leu Arg Tyr Asn His Met Leu Arg Lys Lys Lys Asn Leu Leu Leu Gln 340 345 350 Tyr Asn Ser Gly Glu Asp Leu Ala Val Asn Ile Ile Phe Pro Glu Lys 355 360 365 Ile Asp Met Thr Thr Phe Ser Lys Glu Ala Gly Asp Glu Glu Ile 370 375 380 <210> 3 <211> 5 <212> PRT <213> Mouse (Mus musculus) <400> 3 Asp Thr Tyr Ile His 1 5 <210> 4 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 4 Arg Ile Asp Pro Pro Asn Gly Asn Leu Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Gly <210> 5 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 5 Ser Asp Gly Tyr Ser Phe Ala Tyr 1 5 <210> 6 <211> 16 <212> PRT <213> Mouse (Mus musculus) <400> 6 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr 1 5 10 15 <210> 7 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 7 Arg Met Ser Asn Leu Ala Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 8 Met Gln His Val Glu Tyr Pro Phe Thr 1 5 <210> 9 <211> 5 <212> PRT <213> Mouse (Mus musculus) <400> 9 Asp Tyr Ala Met His 1 5 <210> 10 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 10 Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 11 <211> 12 <212> PRT <213> Mouse (Mus musculus) <400> 11 Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 12 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 12 Lys Ser Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 13 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 13 Phe Ala Ser Thr Arg Glu Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 14 Gln Gln His Tyr Ser Thr Pro Pro Thr 1 5 <210> 15 <211> 354 <212> DNA <213> mouse(Mus musculus) <220> <221> CDS <222> (1)..(354) <400> 15 gag gtt cag ctg cag cag tct ggg gca gag ctt gtg aag cca ggg gcc 48 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 tca gtc aag ttg tcc tgc aca gct tct ggc ttc aac att aga gac acc 96 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Arg Asp Thr 20 25 30 tat ata cac tgg gtg aag cag agg cct gag cag ggc ctg gag tgg att 144 Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 gga agg att gat cct ccg aat ggt aat ctt aaa tat gac ccg aag ttc 192 Gly Arg Ile Asp Pro Pro Asn Gly Asn Leu Lys Tyr Asp Pro Lys Phe 50 55 60 CAG GGC AAG GCC ACT ATA ACA GCA GAC ACA TCC TCC AAC ACA GCC TAC 240 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 CTG CAG TTC AGC AGC CTG ACA TCT GAG GAC ACT GCC GTC TAT TAC TGT 288 Leu Gln Phe Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 GCA AGG TCT GAT GGT TAC TCC TTT GCT TAC TGG GGC CAA GGG ACT CTG 336 Ala Arg Ser Asp Gly Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 GTC ACT GTC TCT GCA GCC 354 Val Thr Val Ser Ala Ala 115 <210> 16 <211> 118 <212> PRT <213> Mus musculus <400> 16 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Arg Asp Thr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Pro Asn Gly Asn Leu Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Phe Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala Ala 115 <210> 17 <211> 336 <212> DNA <213> Mus musculus <220> <221> CDS <222> (1)..(336) <400> 17 gat att gtg atg act cag gct gca ccc tct gta cct gtc act cct gga 48 Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly 1 5 10 15 gag tca gta tcc atc tcc tgc agg tct agt aag agt ctc ctg cat agt 96 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 aat ggc aac act tac ttg tat tgg ttc ctg cag agg cca ggc cag tct 144 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 cct cag ctc ctg ata tat cgg atg tcc aac ctt gcc tca gga gtc cca 192 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 gac agg ttc agt ggc agt ggg tca gga act gct ttc aca ctg aga atc 240 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70 75 80 agt aga gtg gag gct gag gat gtg ggt gtt tat tac tgt atg caa cat 288 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 gta gaa tat cca ttc acg ttc ggc tcg ggg aca aag ttg gaa ata aaa 336 Val Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 18 <211> 112 <212> PRT <213> Mouse (Mus musculus) <400> 18 Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Val Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 19 <211> 363 <212> DNA <213> Mouse (Mus musculus) <220> <221> CDS <222> (1)..(363) <400> 19 cag gtc cag ctg cag cag tct ggg cct gag ctg gtg agg cct ggg gtc 48 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val 1 5 10 15 tca gtg aag att tcc tgc aag ggt tcc ggc tac aca ttc act gat tat Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg aag cag agt cat gca aag agt cta gag tgg att 144 Only Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Serving Thr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gta gac aaa tcc tcc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt gcc aga ttg aca tct gag gat tct gcc atc tat tac tgt 288 Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 gca aga gga gga tta cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc tca gtc acc gtc tcc tca 363 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 20 <211> 121 <212> PRT <213> Mouse (Mus musculus) <400> 20 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 21 <211> 339 <212> DNA <213> Mouse (Mus musculus) <220> <221> CDS <222> (1)..(339) <400> 21 gac att gtg atg aca cag tct cca tcc tcc ctg gct atg tca gta gga 48 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly 1 5 10 15 cag aag gtc act atg agc tgc aag tcc agt cag agc ctt tta aat agt 96 Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 agc aat caa aag aac tat ttg gcc tgg tac cag cag aaa cca gga cag 144 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 tct cct aaa ctt ctg gta tac ttt gca tcc act agg gaa tct ggg gtc 192 Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 cct gat cgc ttc ata ggc agt gga tct ggg aca gat ttc act ctt acc 240 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 atc agc agt gtg cag gct gaa gac ctg gca gat tac ttc tgt cag caa 288 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 85 90 95 cat tat agc act cct ccc acg ttc ggt gct ggg acc aag ctg gag ctg 336 His Tyr Ser Thr Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 aaa 339 Lys <210> 22 <211> 113 <212> PRT <213> Mouse (Mus musculus) <400> 22 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly 1 5 10 15 Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 85 90 95 His Tyr Ser Thr Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 23 <211> 351 <212> DNA <213> Artificial sequence <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(351) <400> 23 cag gtt cag ctg gtg cag tct ggg gca gag gtg aag aag cca ggg tcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 tca gtc aag gtc tcc tgc aag gct tct ggc ttc aac att aga gac acc 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Arg Asp Thr 20 25 30 tat ata cac tgg gtg cgg cag gcc cct gga cag ggc ctg gag tgg att 144 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga agg att gat cct ccg aat ggt aat ctt aaa tat gac ccg aag ttc 192 Gly Arg Ile Asp Pro Pro Asn Gly Asn Leu Lys Tyr Asp Pro Lys Phe 50 55 60 cag ggc aag gcc act ata aca gca gac aca tcc acg agc aca gcc tac 240 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gag ttc agc agc ctg aga tct gag gac act gcc gtc tat tac tgt 288 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca agg tct gat ggt tac tcc ttt gct tac tgg ggc caa ggg act ctg 336 Ala Arg Ser Asp Gly Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 gtc act gtc tct tca 351 Val Thr Val Ser Ser 115 <210> twenty four <211> 117 <212> PRT <213> Artificial <220> <223> Synthetic constructs (recombinant proteins) <400> twenty four Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Arg Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Pro Asn Gly Asn Leu Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 25 <211> 351 <212> DNA <213> Artificial sequence <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(351) <400> 25 cag gtt cag ctg gtg cag tct ggg gca gag gtg aag aag cca ggg tcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 tca gtc aag gtc tcc tgc aag gct tct ggc ttc aac att aga gac acc 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Arg Asp Thr 20 25 30 tat ata cac tgg gtg cgg cag gcc cct gga cag ggc ctg gag tgg att 144 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga agg att gat cct ccg aat ggt aat ctt aaa tat gac ccg aag ttc 192 Gly Arg Ile Asp Pro Pro Asn Gly Asn Leu Lys Tyr Asp Pro Lys Phe 50 55 60 cag ggc aag gcc act ata aca gca gac aca tcc acg agc aca gcc tac 240 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gag ctg agc agc ctg aga tct gag gac act gcc gtc tat tac tgt 288 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca agg tct gat ggt tac tcc ttt gct tac tgg ggc caa gga act ctg 336 Ala Arg Ser Asp Gly Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 gtc act gtc tct tca 351 Val Thr Val Ser Ser 115 <210> 26 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct (recombinant protein) <400> 26 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Arg Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Pro Asn Gly Asn Leu Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 27 <211> 336 <212> DNA <213> Artificial sequence <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(336) <400> 27 gat att gtg atg act cag tct cca ctc tct ctg cct gtc act cct gga 48 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 gag ccg gcc tcc atc tcc tgc agg tct agc aag agt ctc ctg cat agt 96 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 aat ggc aac act tac ttg tat tgg ttc ctg cag aag cca ggc cag tct 144 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 cct cag ctc ctg ata tat cgg atg tcc aac ctt gcc tca gga gtc cca 192 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 gac agg ttc agt ggc agt ggg tca gga act gct ttc aca ctg aaa atc 240 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile 65 70 75 80 agt aga gtg gag gct gag gat gtg ggt gtt tat tac tgt atg caa cat 288 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 gta gaa tat cca ttc acg ttc ggc caa ggg aca aag gtg gaa atc aaa 336 Val Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 28 <211> 112 <212> PRT <213> Artificial <220> <223> Synthetic construct (recombinant protein) <400> 28 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Val Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 29 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(363) <400> 29 caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 30 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct (recombinant protein) <400> 30 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 31 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(363) <400> 31 caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 32 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct (recombinant protein) <400> 32 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 33 <211> 363 <212> DNA <213> Artificial <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(363) <400> 33 caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 34 <211> 121 <212> PRT <213> Artificial <220> <223> Synthetic construct (recombinant protein) <400> 34 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 35 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(363) <400> 35 caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Serving Thr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 36 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct (recombinant protein) <400> 36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 37 <211> 363 <212> DNA <213> Artificial <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(363) <400> 37 caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 38 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct (recombinant protein) <400> 38 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 39 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(363) <400> 39 caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 40 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct (Recombinant Protein) <400> 40 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 41 <211> 363 <212> DNA <213> Artificial <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(363) <400> 41 caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 42 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct (recombinant protein) <400> 42 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 43 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(363) <400> 43 caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 44 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct (recombinant protein) <400> 44 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 45 <211> 339 <212> DNA <213> Artificial sequence <220> <223> Recombinant DNA <220> <221> CDS <222> (1)..(339) <400> 45 gac att gtc atg aca cag tct cca gac tcc ctg gct gtg tca ctg gga 48 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 gag agg gcc act atc aac tgc aag tcc agt cag agc ctt ctg aat agt 96 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 agc aat caa aag aac tat ttg gcc tgg tac cag cag aaa cca gga cag 144 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 cct cct aaa ctt ctg gtc tac ttt gca tcc act agg gaa tct ggg gtc 192 Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 cct gat cgc ttc agt ggc agt gga tct ggg aca gat ttc act ctt acc 240 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 atc agc agt ctg cag gct gaa gat gtg gca gtt tac tac tgt cag caa 288 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 cat tat agc act cct ccc aca ttc ggt cag ggg acc aag ctg gag atc 336 His Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 aaa 339 Lys <210> 46 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct (Recombinant Protein) <400> 46 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys

Claims

1. A combination drug for treating hepatocellular carcinoma, comprising: Lenvatinib or a pharmaceutically acceptable salt thereof; and, An antibody against human DLK-1 having anti-tumor activity in vivo or an antibody fragment derived therefrom, The antibody is an antibody in which the amino acid sequences of CDR1 to 3 of the H chain V region are the amino acid sequences shown in SEQ ID NOs: 9 to 11, respectively, and the amino acid sequences of CDR1 to 3 of the L chain V region are the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively. The antibody fragment is an antibody fragment comprising the amino acid sequence shown in SEQ ID NOs. 9 to 11 and the amino acid sequence shown in SEQ ID NOs. 12 to 14; The antibody fragment has the binding activity of human DLK-1 antibody and in vivo anti-tumor activity.

2. The combination drug according to claim 1, wherein The tumor is hepatocellular carcinoma.

3. The combination drug according to claim 1 or 2, wherein: The antibody is a chimeric antibody or a humanized antibody.

4. The combination drug according to claim 1 or 2, wherein The antibody is an antibody in which the amino acid sequence of the H chain V region consists of the amino acid sequence shown in SEQ ID NO: 36, and the amino acid sequence of the L chain V region consists of the amino acid sequence shown in SEQ ID NO: 46, The antibody fragment is an antibody fragment comprising the amino acid sequence shown in sequence number 36 and the amino acid sequence shown in sequence number 46.

5. The combination drug according to claim 1 or 2, wherein: The antibody or antibody fragment is in the form of a complex with a compound having anti-tumor activity and / or cell-killing activity.

6. The combination drug according to claim 1 or 2, wherein: After the administration of the combined drug is completed, the proliferation of cancer cells can be inhibited or the tumor can be reduced or eliminated.

7. Lenvatinib or a pharmaceutically acceptable salt thereof, and An antibody against human DLK-1 having anti-tumor activity in vivo or an antibody fragment derived therefrom, Use for the manufacture of a medicament for treating hepatocellular carcinoma, The antibody is an antibody in which the amino acid sequences of CDR1 to 3 of the H chain V region are the amino acid sequences shown in SEQ ID NOs: 9 to 11, respectively, and the amino acid sequences of CDR1 to 3 of the L chain V region are the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively. The antibody fragment is an antibody fragment comprising the amino acid sequence shown in SEQ ID NOs. 9 to 11 and the amino acid sequence shown in SEQ ID NOs. 12 to 14; The antibody fragment has the binding activity of human DLK-1 antibody and in vivo anti-tumor activity.

8. A kit for treating hepatocellular carcinoma, comprising: Lenvatinib or a pharmaceutically acceptable salt thereof, and An antibody against human DLK-1 having anti-tumor activity in vivo or an antibody fragment derived therefrom, The antibody is an antibody in which the amino acid sequences of CDR1 to 3 of the H chain V region are the amino acid sequences shown in SEQ ID NOs: 9 to 11, respectively, and the amino acid sequences of CDR1 to 3 of the L chain V region are the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively. The antibody fragment is an antibody fragment comprising the amino acid sequence shown in SEQ ID NOs. 9 to 11 and the amino acid sequence shown in SEQ ID NOs. 12 to 14; The antibody fragment has the binding activity of human DLK-1 antibody and in vivo anti-tumor activity.

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