JP2003000249A - ACTIVATION OF proMMP-2 WITH CLAUDINS THROUGH MT-MMPs - Google Patents

Abstract

PROBLEM TO BE SOLVED: To elucidate a gene relating to activity regulation of proMMP-2 activation with MT-MMPs such as MT1-MMP and carry out researches and developments of various biological activities.physiological phenomena as for proMMp-2 activation. SOLUTION: Claudins have a function to accelerate proMMP-2 activation with MT-MMPs such as MT1-MMP and researches and developments of useful medicinal agents, and the like, are enabled through elucidating the function of the same. A mutant claudin protein has functions to suppress and/or inhibit the proMMP-2 activation and is useful as a suppressing and/or inhibiting agent for vascularization and moving, transferring and infiltration of cancer cells.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to claudins (c
laudins) are membrane-type matrix metalloproteases (mem
brane-type matrix metalloproteinases (MT-MMPs) -mediated latent matrix metalloproteinase-2 (proMMP-
2) Concerning promoting activation. The present invention also relates to a substance that inhibits MT-MMPs-mediated proMMP-2 activation by the claudins, particularly a substance that inhibits MT1-MMP-mediated proMMP-2 activation. The present invention inhibits complex formation between the claudins and MT-MMPs and / or proMMP-2, and thus inhibits activation of proMMP-2, resulting in various activations of proMMP-2. To control various physiological and biological processes and drugs therefor. The present invention further focuses on the extracellular domain of claudins, MT-MMPs
In particular, the present invention provides a medicine or therapeutic method for inhibiting invasion and metastasis of cancer based on a proMMP-2 activation regulation mechanism mediated by MT1-MMP.

[0002]

PRIOR ART Matrix metalloproteases (MMP
s) is the extracellular matrix: E
It is a family of zinc-dependent endopeptidases that are thought to be essential for extracellular matrix metabolism by degrading various constituent proteins of CM) and basement membrane components. These enzymes are involved in connective tissue remodeling, such as normal embryo development, bone growth or wound healing, and atherosclerosis, emphysema, rheumatoid arthritis, cancer invasion and metastasis. The involvement in various pathological processes such as is becoming known. To date, many mammalian MMPs have been analyzed at the amino acid level by cDNA cloning. For example, MMP-1 (collagenase);
MMP-2 (gelatinase A); MMP-3 (stromelysin-1); MMP-
7 (matrilysin); MMP-8 (neutrophil collagenase); MM
P-9 (gelatinase B); MMP-10 (stromelysin-2); MMP-11
(stromelysin-3); MMP-12 (macrophage elastase); MM
P-13 (collagenase-3); MMP-14 (MT1-MMP); MMP-15 (MT
2-MMP); MMP-16 (MT3-MMP); MMP-17 (MT4-MMP); MMP-1
9; MMP-20 (enamelysin); MMP-24 (MT5-MMP); MMP-25
(MT6-MMP) etc. These MMPs, depending on their primary structure, substrate specificity and cell distribution, have at least four subfamilies: collagenase, gelatinase, stromelysin and membrane-type matrix metalloproteases (M
T-MMPs), of which the MT-MMPs subfamily is the most recently reported subclass of MMPs.For example, MT1-MMP, MT2-MMP, MT3-MMP, MT4
-MMP, MT5-MMP, MT6-MMP and the like, which are type I membrane proteins having a single transmembrane region behind the hemopexin domain characteristic of many MMPs.

When cells move, infiltrate, and metastasize in tissues, the decomposition of extracellular matrix (ECM) surrounding them is an essential step. MMPs, which play a central role in this step, are a group of enzymes called MMPs, and among them, MT1-MMPs expressed on the cell membrane surface are important for their role in cancer migration, invasion, metastasis, and angiogenesis. MT1-MMP
Is a membrane-type matrix metalloprotease-1 (membrane
-type 1 matrix metalloproteinase: MT1-MMP) or
An enzyme (MEROPS ID: M10.014) also called MMP-14, which is a gene occupying its chromosomal locus 14q11-q12 in humans (C. Mignon et al., Genomics, 28: pp.360-361).
(1995)) and DNA
The successful cloning and expression of the recombinant protein confirmed its existence and clarified the detailed structure and characteristics (H. Sato et al., Natur.
e, 370: pp.61-65 (1994); T. Takino et al., Gene, 1
55: pp.293-298 (1995); JP-A-7-203961; JP-A-7-303482; GenBank ^TM accession number: D26
512). The presence of MT1-MMP has been confirmed in dogs, goats, rabbits, boars, rats, etc. as well as humans. The human MT1-MMP cDNA encodes 582 amino acid residues.
(EMBL accession No. D26512, E09720 & E 10297; SWISS
-PROT: P50281), the structure of which is the signal peptide followed by the propeptide domain, stromelysin-3 (stromelysi
n-3) Insertion sequence consisting of 10 unique amino acid residues (a sequence that may be a recognition site for a furin-like enzyme), a core enzyme that has a potential zinc-binding site It consists of domains, hinge domains, hemopexin-like domains, and transmembrane (TM) domains.

To date, MT1-MMP is the same MMP member and basement membrane degrading enzyme gelatinase A (MMP-).
2) activating latent type (progelatinase A / 72kDa type IV collagenase: proMMP-2), and MT1-MMP itself also has type I, type II and type III collagen, fibronectin,
Various ECs such as laminin, vitronectin and aggrecan
It is known to decompose M molecules. Also MT1-MMP
Was also shown to promote the process of tumor invasion and metastasis (S
eiki, M., Apmis, 107, 137-143 (1999); Sato, H. et a
L., Nature, 370, 61-65 (1994)). MT1-MMP is also pro
MMP-2 (Sato, H. et al., Nature, 370, 61-65 (1994))
And procollagenase-3 (proMMP-13) (Knauper, V. et a
l., J. Biol. Chem., 271, 17124-17131 (1996)) to activate other MMPs. Thus, MT1-MMP expression may be involved in the initiation of diverse proteolytic enzyme cascades on the cell surface, and MT1-MMP induces cancer cell invasion and metastasis (Seiki, M., Apmis, 107). , 137-143 (1999); Sa
to, H. et al., Nature, 370, 61-65 (1994)) as well as angiogenesis (Hiraoka, N. et al., Cell 95, 365-77 (19
98); Zhou, Z. et al., Proc. Natl. Acad. Sci. USA,
97, 4052-4057 (2000)) and skeletal development (Zhou, Z. et al.,
Proc. Natl. Acad. Sci. USA, 97, 4052-4057 (2000);
It has also been shown to be involved in other physiological processes such as Holmbeck, K. et al., Cell, 99, 81-92 (1999)). Thus MT1-MMP appears to be a necessary tool for physiological and pathological cell invasion in tissues, and therefore it is important to elucidate the mechanism of MT1-MMP action at the cell surface. .

It has been shown that MT1-MMP activates proMMP-2 and promotes cancer cell invasion by directly destroying the extracellular matrix. Cell surface activation of proMMP-2 by MT1-MMP appears to be an important step in tumor invasion. Because, MMP-2 decomposes type IV collagen and laminin, which are the main components of the basement membrane (S
tetler-Stevenson, WG et al., Annu. Rev. Cell Bi
ol., 9, 541-73 (1993)). Occurrence of active MMP-2 also correlates well with tumor invasiveness (Tokuraku, M. et al., Int.
J. Cancer, 64, 355-359 (1995); Nomura, H. et al.,
Cancer Res., 55, 3263-3266 (1995)). By the way, it is considered that TIMP-2, which forms a complex with proMMP-2 preferentially in the MT1-MMP-mediated proMMP-2 activation process, has played a central role. In this model, activated MT1-MMP first binds to TIMP-2 via its catalytic domain. In this way, soluble TIMP-2 is bound on the cell surface. This TIMP-2-MT1-MMP complex continues to act as a receptor for proMMP-2,
The interaction between the C-terminal domain of proMMP-2 and the C-terminal domain of TIMP-2 forms a ternary complex. proMMP-2
Propeptide of MT1-adjacent MT1-unbound TIMP-2
Asn ⁶⁶ -Leu ⁶⁷ (SWISS-PROT accession No.
P08253) to form an active intermediate. this
The MMP-2 intermediate, when present at sufficiently high concentrations on the cell surface, is further processed into its fully active form by intermolecular auto-cleavage. This first step of activation is blocked when TIMP-2 is present in excess relative to MT1-MMP. In order to achieve localized enrichment of the ternary complex, it is required to localize to a unique portion on the membrane.

[0006] In multicellular organisms such as mammals including humans, information on adhesion with adjacent cells includes cell proliferation,
It is deeply involved in differentiation and migration, further migration, invasion and metastasis of cells such as cancer, and regulation and maintenance of life phenomena including pathological phenomena such as inflammation. The intercellular adhesion molecules involved in such adhesion are known to assemble on the cell surface to form a specially differentiated membrane region for adhesion, which is called the intercellular adhesion device, Primarily gap junction (GJ), adherens junction (AJ), d
It is classified into four categories, esmosome and tight junction (TJ). What was discovered as one of the constituent membrane proteins that make up the TJ is a group of claudins. The Claudin family is a major component of the TJ chain, which is paracellular sealant (barrier function) and membrane domain differentiation (enclosure function) in epithelial and endothelial cell sheets.
Are directly involved in. To date, 18 claudin families have been identified. Examples of the claudins family include claudin-1 (claudin-1), claudin-2 (claudin-2), claudin-3 (claudin-3) and claudin-5 (claudin-5), Claudin-6 (cla
udin-6), claudin-7 (claudin-7), claudin
-8 (claudin-8) has been reported (Japanese Patent Laid-Open No. 2000-32984
Publication: Furuse, M., et al., J. Cell Biol., 141: 15
39-1550 (1998) and Furuse, M., et al., J. Cell Bi
ol., 147: 891-903 (1999)). However, the physiological significance of the interaction between claudin, MT-MMPs and proMMP-2 has not been known so far.

[0007]

Regarding the relationship between MMPs and / or membrane-type MMPs and cancer so far, for example, activation of progelatinase A (Okada et al., Eur. J. Bioche
m., 194, pp721-730, 1990), and MT1-MMP activation of MMP-2 (Sato et al., Nature, 370, pp61-65, 1994). Is still unknown. It has activity against various constituent proteins of extracellular matrix and basement membrane components
MMPs not only affect various pathological symptoms and onset due to their increased activity, but also metastasis of cancer etc.
It has been suggested that it is involved in invasion and further in angiogenesis around the cancer tissue. Therefore, it is required to elucidate what kinds of genes and proteins are involved in the regulation of MMPs activity, and by what mechanism they are involved in the regulation of MMPs activity. Therefore, in order to search for measures that control the migration, invasion, and metastasis of cells such as cancer, it is necessary to advance the factors that control the activation of proMMP-2 by MT1-MMP and further research on it. There is.

[0008]

[Means for Solving the Problems] The present inventors have proposed MT-MMPs.
We constructed a screening system for genes involved in the regulation of activation of latent MMP-2 (proMMP-2) through , MT-MMPs mediated proMMP-2 activation-promoting activity, and further, a modified extracellular domain of claudin is the MT-MMPs mediated proMMP
The present invention has been completed by finding that the inhibitory activity against MP-2 activation is exhibited.

That is, the present invention relates to [1] a method for activating proMMP-2 via MT-MMPs by one selected from the claudin family; [2] one selected from the claudin family, claudin -1, Claudin-2, Claudin-
3, or Claudin-5, the method according to [1] above; [3] The above [1] or [1] characterized by MT1-MMP-mediated activation of proMMP-2. [2] The method as described above; [4] (1) Inhibiting complex formation between one selected from the claudin family and (2) one selected from MT-MMPs and proMMP-2, proMMP-2 activation inhibitor; [5] (1) characterized by inhibiting complex formation between one selected from claudin family and (2) selected from MT1-MMP and proMMP-2 [4] described above
proMMP-2 activation inhibitor;

[6] (i) inhibits the formation of a complex between one selected from the claudin family and one selected from MT-MMPs to inhibit the activation of proMMP-2, or (ii) Selected from the claudin family
An agent for inhibiting angiogenesis, cell migration, invasion and / or metastasis, characterized by inhibiting complex formation with proMMP-2 to inhibit activation of proMMP-2; [7] The cell is a cancer cell [8] The complex according to [6] above, wherein the complex is (1) selected from the claudin family and (2) selected from MT1-MMP and proMMP-2. An agent according to the above [6] or [7], which is a complex of

[9] The complex formation is performed at least between extracellular domains of those selected from the claudin family, [4] to
[10] The agent according to any one of [8]; [10] (1) Inhibiting complex formation between one selected from the claudin family and (2) one selected from MT-MMPs and proMMP-2 A method for inhibiting proMMP-2 activation, which is characterized by: [11] (1) Complex formation between one selected from the claudin family and (2) selected from MT1-MMP and proMMP-2 The method according to the above [10], which comprises inhibiting;

[12] (i) inhibits the formation of a complex between one selected from the claudin family and one selected from MT-MMPs to inhibit the activation of proMMP-2, or (ii) Angiogenesis, cell migration, which is characterized by inhibiting the formation of a complex selected from the claudin family and proMMP-2 to inhibit the activation of proMMP-2,
A method for preventing invasion and / or metastasis; [13] The method according to [12] above, wherein the cell is a cancer cell; [14] The complex is selected from the (1) claudin family [12] above, which is a complex of a substance selected from the group consisting of (2) MT1-MMP and proMMP-2
Or the method of [13]; [15] The complex formation is carried out at least between the extracellular domain of the claudin family and the above-mentioned [10]-
The method according to any one of [14];

[16] (1) A substance having an activity of inhibiting complex formation between a substance selected from the claudin family and (2) a substance selected from MT-MMPs and proMMP-2; [17] complex The body is a complex with (a) one selected from the claudin family present on the cell surface or (b) one selected from MT-MMPs present on the cell surface, [16] ] [18] (A) A claudin selected from the claudin family and having a mutation in its extracellular domain region, a derivative thereof or an analogue thereof, or an activity substantially equivalent thereto. (B) a peptide fragment selected from the claudin family and corresponding to a part of the peptide corresponding to its extracellular domain region, or an activity substantially equivalent thereto. [16] or [17] above, wherein the derivative is selected from the group consisting of an antibody against the extracellular domain region of the derivative selected from the claudin family or a derivative thereof; Substance of

[19] (A) The mutation in the extracellular domain region of the one selected from the claudin family is selected from the group consisting of deletion, substitution and insertion of amino acid residues; (B) ( i) The peptide corresponding to the extracellular domain region is the amino acid sequence Asp ^{38 to} Phe ⁶⁷ of human claudin-1 shown in SEQ ID NO: 1 in the sequence listing.
Amino acid sequence of human claudin-1 corresponding to the peptide Val ^{55 to} Cys ⁶⁴
Peptide corresponding to the amino acid sequence of human claudin-1 Asn ¹⁴² ~ Val
Selected from the group consisting of peptides corresponding to ¹⁵⁵ and (ii)
The peptide having at least 3 or more consecutive amino acid residues among the peptides; or (C) (i) the peptide corresponding to the extracellular domain region is the human claudin described in SEQ ID NO: 1 of the Sequence Listing. -1 amino acid sequence Asp ^{38 to} Phe ⁶⁷
Amino acid sequence of human claudin-1 corresponding to the peptide Val ^{55 to} Cys ⁶⁴
Peptide corresponding to the amino acid sequence of human claudin-1 Asn ¹⁴² ~ Val
Selected from the group consisting of peptides corresponding to ¹⁵⁵ and (ii)
The substance according to any one of [16] to [18] above, which is an antibody against one having at least three consecutive amino acid residues among the peptides.

[20] (i) a nucleic acid encoding the one described in (A) of [18] above, (ii) a part of the peptide corresponding to the extracellular domain region described in (B) of [18] above A nucleic acid encoding a corresponding peptide fragment, (iii) a nucleic acid capable of hybridizing with the nucleic acid of (i) above, and (iv)
A nucleic acid selected from the group consisting of nucleic acids having substantially the same activity as any one of the above (i) to (iii); [21] (i) [19] ] (A) a mutant claudin, a derivative thereof or an analog thereof, or a nucleic acid encoding the same having an activity substantially equivalent thereto, (ii) the peptide fragment described in (B) of [19] above. Encoding nucleic acid, (iii) a nucleic acid capable of hybridizing with the nucleic acid of (i) above, and (iv) a nucleic acid having substantially the same activity as any one of the nucleic acids of (i) to (iii) above [22] A nucleic acid according to the above [20], which is selected from the group consisting of: [22] A mutant lacking the amino acid residues at positions 38-67 of human claudin-1 (M1Δ38-67). ), Human Claudin-1
Of the mutant (M2Δ55-6
4), a mutant lacking amino acid residues 142-155 of human claudin-1 (M3Δ142-155), human claudin-1
A mutant in which the cysteine residue at position 54 was replaced with a serine residue (M
4C54S), a mutant in which the cysteine residue at position 64 of human claudin-1 was replaced with a serine residue (M5C64S), a mutant in which the PDZ motif on the C-terminal side of human claudin-1 was destroyed (M6Δ
YV) and a mutant selected from the group consisting of the amino acid residues 1-101 at the N-terminal side of human claudin-1 (M7Δ1-101), or an activity substantially equivalent thereto. [20] which is characterized by encoding a code having
Or the nucleic acid according to [21];

[23] A vector containing the nucleic acid according to any one of the above [20] to [22]; [24] The nucleic acid according to any one of the above [20] to [22] A transformant comprising the vector according to [23] above; [25] containing the nucleic acid according to any one of [20] to [22] above or the vector according to [23] above. [26] The above-mentioned [4]-

[9] A medicament or a veterinary medicament containing any one of [9] or [16] to [24] above; [27] Due to proMMP-2 activation The drug or veterinary drug according to the above [26], which is for treating and / or preventing a disease or a pathological condition; [28] Angiogenesis, cell migration, invasion and / or metastasis The drug or veterinary drug according to the above [26], which is for suppressing and / or blocking; [29] The drug according to the above [28], wherein the cell is a cancer cell or Veterinary medicine;

[30] (1) Inhibition of activation of proMMP-2 by using as an index a complex formation of one selected from the claudin family and (2) one selected from MT-MMPs and proMMP-2 [31] (1) Inhibiting complex formation between a substance selected from the claudin family and (2) a substance selected from MT1-MMP and proMMP-2 [32] The method described in [30] above, wherein: [32] (1) expression of claudin family, (2) selection of MT-MMPs, and (3) expression of proMMP-2 The method of [30] or [31] above, which comprises using a cell line of the present invention; [33] ProMMP-2 obtained by the method of any one of [30] to [32] above A substance that inhibits activation; [34] (i) Selected from the claudin family Selected from the MT-MMPs to inhibit the formation of proMMP-2 activation, or (ii) those selected from the claudin family and proMMP-2. A screening method characterized by screening a substance having an activity of inhibiting angiogenesis, cell migration, invasion and / or metastasis by using the inhibition of complex formation and proMMP-2 activation as an index [35] characterized by using a cell line expressing (1) one selected from the claudin family, (2) one selected from MT-MMPs, and (3) proMMP-2 The method described in [34] above;

[36] A substance having angiogenesis, cell migration, invasion and / or metastasis inhibitory activity obtained by the method according to [34] or [35] above; [37] Screening for inhibition of proMMP-2 activation In the presence of (i) (1) claudin family, (2) selected from MT-MMPs, and (3) proMMP-2 in the presence of (ii) activation inhibition of proMMP-2. A substance which inhibits the activation of proMMP-2, which comprises comparing the degree of activation of proMMP-2 in the presence of a candidate compound or (iii) the inhibition of activation of said proMMP-2. Screening method: [38] using a cell line expressing (1) one selected from the claudin family, (2) one selected from MT-MMPs, and (3) proMMP-2 [39] (1) Selected from the claudin family Ones and (2) MT1-MMP and (3) above [37], wherein the to be screened in the presence of proMMP-2 or [3
8) The method described in [8]; and [40] The substance which inhibits the activation of proMMP-2 obtained by the method described in any one of [37] to [39] above is provided.

Other objects, features, excellences, and aspects of the present invention will be apparent to those skilled in the art from the following description. However, it is understood that the description of the present specification, including the description below and the description of specific examples and the like, shows the preferred embodiments of the present invention and is provided only for the purpose of explanation. I want to. Various changes and / or alterations (or modifications) within the spirit and scope of the invention disclosed herein can be made by those of ordinary skill in the art based on knowledge from the following description and other portions of the specification. Would be readily apparent. All patent and literature references cited in this specification are cited for the purpose of illustration and are to be construed as part of this specification, the contents of which are included herein. is there. In this specification,
The term “and / or” means that there are both (1) a merged connection relationship and (2) a selective connection relationship, eg in the case of “treatment and / or prevention” (1) It is used to mean both treatment and prevention and (2) treatment or prevention. In other cases, the term “and / or” is similarly used to include both (1) merged connection and (2) selective connection.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, proMMP-2 mediated by MT-MMPs by a protein belonging to the claudin family
An activation method, a method for inhibiting the activation, a substance and a drug having the inhibitory activity thereof are provided, and a method for screening such an inhibitory active substance and a drug used therefor are also provided. In particular, the extracellular domain region of claudin plays an important role in MT1-MMP-mediated activation of proMMP-2.For example, a protein having a mutation in the extracellular domain region of claudin is expressed in vivo and in vitro.
Shows promising activity for MT1-MMP-mediated activation of proMMP-2 and is promising as an inhibitor or drug. Based on this fact, we will develop an inhibitor of proMMP-2 activation by various claudins. To enable that. The present invention, on the one hand,
Complex of claudin and MT-MMPs or claudin
The present invention provides a method for monitoring the formation of a complex or the like consisting of phenotype and proMMP-2. It also provides a way for gene therapy. The present invention relates to an antibody that specifically recognizes the extracellular domain region of claudins, for example, a monoclonal antibody, and an immunological measurement reagent or drug using the antibody,
Furthermore, various measurement methods are also provided.

According to the present invention, a variant derived from one of the claudins family and having at least 60% homology with the polypeptide having proMMP-2 activation inhibitory activity or the amino acid sequence of said variant claudin polypeptide. And a peptide or salt thereof having proMMP-2 activation inhibitory activity or equivalent antigenicity, a partial peptide characteristic of the polypeptide or salt thereof, a gene encoding them, for example, a gene such as DNA or RNA. A vector or plasmid containing such that it can be manipulated by gene recombination technology, a host cell transformed with such a vector, and a transgenic animal such as a transgenic mouse expressing the gene, A method of culturing a transformed cell to produce the polypeptide or a salt thereof, An antibody obtained by using the polypeptide or a salt thereof or a partial peptide characteristic of the polypeptide or a salt thereof, particularly a monoclonal antibody, a hybridoma cell producing the antibody, the predetermined coding gene, For example, a measuring / diagnosing means and a reagent using DNA or RNA as a probe or using the antibody are provided. Further provided is the use of the active ingredients as disclosed and described herein,
For example, a drug or a reagent containing the active ingredient is provided, and a method for treating and / or preventing a disease, a disease or an abnormal condition using the active ingredient, and a screening method are provided. The mutant claudin polypeptide is preferably one in which the extracellular domain region of claudin has been mutated.

As used herein, the term "polypeptide" may refer to any polypeptide as described below. The basic structure of polypeptides is well known and is described in numerous reference books and other publications in the art. In view of these, the term “polypeptide” as used herein means any peptide or any protein containing two or more amino acids which are linked to each other by peptide bonds or modified peptide bonds. To do. As used herein, the term “polypeptide” is commonly referred to in the art as a short chain, also commonly referred to as a peptide, oligopeptide or peptide oligomer, and as a protein, which is known in many forms. It may mean both of the long chains mentioned. Polypeptides often contain amino acids other than the 20 amino acids normally present (the 20 naturally occurring amino acids: or the amino acids that are encoded by the gene). May be. Polypeptides may also be subjected to natural processes such as processing and other alterations (or modifications) after translation of many amino acid residues, including terminal amino acid residues, as well as chemistry well known to those skilled in the art. It will be understood that the above-mentioned polypeptide can be modified (modified) also by a technical modification technique. There are many known forms of alterations (modifications) to be made to the polypeptide, which are described in detail in basic reference books and more detailed papers in this field as well as in many research literatures. However, these are well known to those skilled in the art. Some particularly routine modifications include, for example, glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-
Ribosylation and the like, for example, TE Creighton, Pro
teins-Structure and Molecular Properties, Second E
dition, WH Freeman and Company, New York, (199
3); BCJohnson (Ed.), Posttranslational Covalent M
odification of Proteins, Academic Press, New York,
(1983) (Wold, F., "Posttranslational Protein Modi
fications: Perspective and Prospects ", pp.1-12); S
eifter et al., "Analysis for Protein Modifications
and nonprotein cofactors ", Meth. Enzymol., 182: 6
26-646 (1990); Rattan et al., "Protein Synthesis:
Posttranslational Modification and Aging ", Ann. N.
See the description of Y. Acad. Sci., 663: p.48-62 (1992) and the like.

In the present specification, the term "homology" refers to a residue of each amino acid that constitutes a chain between two chains in a polypeptide sequence (or amino acid sequence) or a polynucleotide sequence (or base sequence). Meaning the amount (number) of groups or groups that can be determined to be identical in the mutual correspondence relationship of the bases, and the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences To do. Homology can be easily calculated. There are many known methods for measuring homology between two polynucleotide or polypeptide sequences, the term "homology".
The term (also referred to as “identity”) is well known to those of skill in the art (eg, Lesk, AM (Ed.), Computational
Molecular Biology, Oxford University Press, New Y
ork, (1988); Smith, DW (Ed.), Biocomputing: Info
rmatics and Genome Projects, Academic Press, New Y
ork, (1993); Grifin, AM & Grifin, HG (Ed.),
Computer Analysis of Sequence Data: Part I, Human
Press, New Jersey, (1994); von Heinje, G., Sequence
Analysis in Molecular Biology, Academic Press, New
York, (1987); Gribskov, M. & Devereux, J. (Ed.),
Sequence Analysis Primer, M-Stockton Press, New Yo
rk, (1991) etc.). A general method used to measure the homology of two sequences is Martin, J. Bishop (Ed.), G.
uide to Huge Computers, Academic Press, San Diego,
(1994); Carillo, H. & Lipman, D., SIAM J. Applied
Math., 48: 1073 (1988) and the like, but are not limited thereto. Preferred methods to measure homology include those designed to give the largest match between the two sequences tested. Such methods include those assembled as a computer program. A preferred computer program method for determining homology between two sequences includes the GCG program package (Devereux, J. et al., Nucleic Acids Resea
rch, 12 (1): 387 (1984)), BLASTP, BLASTN, FASTA (A
tschul, SF et al., J. Molec. Biol., 215: 403 (1
990)) and the like, but is not limited thereto, and a method known in the art can be used,
A commercially available product can be used.

In the present specification, "polymerase chain.
Reaction (Polymerase Chain Reaction) "or" PC
“R 2” generally refers to methods such as those described in US Pat. No. 4,683,195 and the like, eg, for in vitro enzymatic amplification of a desired nucleotide sequence. In general, the PCR method involves repeating a cycle for primer extension synthesis using two oligonucleotide primers capable of preferentially hybridizing with a template nucleic acid.
Typically, the primer used in the PCR method can be a primer complementary to the nucleotide sequence to be amplified inside the template, and for example, the nucleotide sequence to be amplified and the complementary nucleotides at both ends thereof can be used. Those that are target or are adjacent to the nucleotide sequence to be amplified can be preferably used. The PCR reaction is
It can be carried out by a method known in the art or a method substantially similar thereto or a modified method, and for example, R. Saik
i, et al., Science, 230: 1350, 1985; R. Saiki, et a
l., Science, 239: 487, 1988; HA Erliched., PCR
Technology, Stockton Press, 1989; DM Glover e
t al. ed., "DNA Cloning", 2nd ed., Vol. 1, (The Pra
ctical Approach Series), IRLPress, Oxford Universi
ty Press (1995); MA Innis et al. ed., "PCRProt
ocols: a guide to methods and applications ", Acade
mic Press, New York (1990)); MJ McPherson, P. Q
uirke and GR Taylor (Ed.), PCR: a practical app
roach, IRL Press, Oxford (1991); MA Frohman et
al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002 (19
88) etc. or modified it,
It can be performed according to a modified method. In addition, the PCR method can be performed using a commercially available kit suitable for the PCR method, or can be performed according to the protocol disclosed by the kit manufacturer or the kit vendor.

In a typical case, for example, a mold (for example,
DNA synthesized using mRNA as a template; 1st strand DNA) and a primer designed based on the gene
Reaction buffer (included with Taq DNA Polymerase)
, DNTPs (deoxynucleoside triphosphate dATP, dGTP,
dCTP, dTTP mixture), Taq DNA polymerase and deionized distilled water. Mix the mixture with, for example, GeneAmp
The cycle is repeated 25 to 60 times under general PCR cycle conditions using an automatic thermal cycler such as 2400 PCR system, Perkin-Elmer / Cetus, etc. Can be PCR cycle conditions include, for example, denaturation 90 to 95 ° C.
5 to 100 seconds, annealing 40 to 60 ° C 5 to 150 seconds, extension 65
~ 75 ° C 30-300 seconds cycle, preferably denaturation 94 ° C
A cycle of 15 seconds, annealing at 58 ° C for 15 seconds and extension at 72 ° C for 45 seconds can be mentioned. The annealing reaction temperature and time can be appropriately selected by experiments, and the denaturation reaction and extension reaction times are also expected. The appropriate value can be selected according to the chain length of the PCR product. The reaction temperature for annealing is usually that of the hybrid between the primer and template DNA.
It is preferable to change according to the Tm value. The extension reaction time is usually about 1 minute per chain length of 1000 bp, but a shorter time may be selected in some cases.

The obtained PCR product is usually subjected to 1 to 2% agarose gel electrophoresis and cut out from the gel as a specific band, and the DNA is extracted using a commercially available extraction kit such as gene clean kit (Bio 101). To do. Extracted
Cleave the DNA with an appropriate restriction enzyme, purify if necessary, and further phosphorylate the 5'end with T4 polynucleotide kinase if necessary, then pUC18 or other pUC
Ligation into an appropriate plasmid vector such as a system vector, and transformation of an appropriate competent cell.
A cDNA library can also be constructed based on the prepared DNA fragment by using a phage vector or a plasmid vector. For transformation of host cells such as E. coli, for example, calcium method, rubidium / calcium method, calcium / manganese method, TFB high efficiency method, FSB
Freezing competent cell method, rapid colony method, electroporation, or any other method known in the art or a method substantially similar thereto can be used (D. Hanaha
n, J. Mol. Biol., 166: 557, 1983). The nucleotide sequence of the cloned PCR product is analyzed.

In the present specification, "claudin"
Is found as one of the constituent membrane proteins that make up the group of TJs described above, and its general structure is four transmembrane domain regions, two extracellular domain regions, and N -And C- consists of the intracellular domain region on the C-terminal side. Examples of proteins belonging to the claudin family include claudin-1 (claudin-1), -2,
-3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -14, -1
5, -16, -17, -18, -19, -20, -21, -22, etc. are known (J. Cell Biol., 141: 1539-1550 (1998); Proc.
Natl. Acad. Sci. USA, 96: 511-516 (1999); Gene, 2
26 (2): 285-295 (1999); Hum. Genet., 107 (3): 249-256.
(2000); J. Biol. Chem., 272 (42): 26652-26658 (199
7); Genomics, 42 (2): 245-251 (1997); Genomics, 46
(3): 443-449 (1997); NCBI Inter Net Data Bases, etc.)
. Examples of claudins include those derived from mammals, and preferably those derived from humans. Claudin-1, a protein belonging to the human claudin family
For -2, -3, -5, etc., data relating to MT1-MMP-mediated proMMP-2 activation have been obtained, as described below.

Those selected from the claudin family have an activity of promoting MT-MMPs-mediated proMMP-2 activation, and in particular, an activity of promoting MT1-MMP-mediated proMMP-2 activation. Have. This proMMP-2 activation promoting activity includes
For example, one selected from the claudin family
Formation of a complex with a selection of MT-MMPs,
Selected from claudin family and proMMP-2
Experimental data supporting the involvement of the formation of a complex with and the like have been obtained. The proMMP-2 activation-promoting activity is predicted to be based on the amino acid sequence portion of the extracellular domain region of claudin, and a polypeptide having these amino acid sequences or an amino acid sequence substantially equivalent thereto is MT-MMPs. Selected from or proMMP-2
It is considered that they form a complex with and exhibit the ability to activate proMMP-2. These means that the mutagenized extracellular domain region of claudin has inhibitory activity against proMMP-2 activation. Furthermore, clau
A peptide fragment corresponding to all or part of a peptide corresponding to the extracellular domain region of din, a derivative having substantially the same activity as those, or an analog thereof is claudi
antagonizes the function of the extracellular domain region of n, proM
It may be considered to mean having an inhibitory activity on MP-2 activation.

[0028] For example, human claudin-1 is
A cell having the amino acid sequence represented by SEQ ID NO: 1
The outer domain regions are each the amino acid sequence Gln.²⁹~ Arg⁸¹,
 Thr ¹³⁷~ Gln¹⁶³it is conceivable that. Therefore, the human
In the case of Rhodin-1, of the extracellular domain region
In which the mutations were introduced as described below.
It may be a variant, an analog, a derivative,
Amino acid sequence corresponding to the main region Asp³⁸~ Phe⁶⁷, Val
⁵⁵~ Cys⁶⁴And Asn¹⁴²~ Val¹⁵⁵Selected from the group consisting of
Peptide or a peptide fragment corresponding to a part of the peptide
Pieces or derivatives having substantially the same activity
Alternatively, they may be (1) proMMP-2 active.
Sexual inhibition activity, (2) (a) Claudin family
Selected from (b) MT-MMPs and proMMP-2
The activity of inhibiting the formation of a complex with, especially (a)
Selected from claudin family and (b) MT1-M
Inhibits complex formation with those selected from MP and proMMP-2.
Harmful activity, selected from the (3) (i) claudin family
Formation of selected compounds from MT-MMPs
To inhibit activation of proMMP-2, or (i
i) selected from claudin family and proMMP
-2 inhibits complex formation with proMMP-2 activation.
Suppresses changes in cells and tissues associated with proMMP-2 activation.
/ Or inhibition or physiological changes associated with proMMP-2 activation
And / or inhibiting biological changes and / or blood
Activities that prevent tube formation, cell migration, invasion and / or metastasis
Indicates sex, etc. If it has the above properties,
At least 60% higher homology with the amino acid sequence of zinal
, Preferably 70% or more homology, more preferably 80
% Or more homology, and preferably 85% or more homology,
Preferably 90% or more homology, more preferably 95%
Have at least the above homology, particularly preferably at least 97% homology
Or substantially equivalent organisms such as equivalent antigenicity
All having amino acid sequences with biological activity are listed.
You may be kicked.

The mutant claudin, claudin extracellular domain peptide fragment and the like are not particularly limited as long as they have an activity of inhibiting proMMP-2 activation, but the activity of inhibiting complex formation as described above. And preferably MT1-present on the cell surface
Those having an activity of inhibiting MMP-mediated activation of proMMP-2 can be mentioned. As a typical one, one having a mutation in the extracellular domain region of human claudin-1, for example, a mutant lacking the amino acid residues at positions 38-67 of human claudin-1 (M1Δ38-67), A mutant lacking the amino acid residues 55-64 of human claudin-1 (M2Δ55-64), a mutant lacking the amino acid residues 142-155 of human claudin-1 (M3Δ142-155) ), A mutant in which the cysteine residue at position 54 of human claudin-1 was replaced with a serine residue (M4C54S), and a mutant in which the cysteine residue at position 64 of human claudin-1 was replaced with a serine residue (M5C64S). ), A mutant in which the PDZ motif at the C-terminal side of human claudin-1 was disrupted (M6ΔYV) and a mutant in which the amino acid residue at position 1-101 at the N-terminal side of human claudin-1 was deleted ( M7Δ1-101), selected from the group consisting of amino acid sequence ^{Asp 38 ~Phe 67, Val 55 ~Cys} 64 and Asn ¹⁴² ~Val ¹⁵⁵ corresponding to the extracellular domain region What was or derivative or its analog having a peptide fragment or their substantially equivalent activity equivalent to a part of the peptide include, but are not limited thereto. As the peptide fragment, consecutive amino acid residues of 5 or more, preferably 10 or more, preferably 20 or more, more preferably 30 or more, more preferably 40 or more, and also preferably
50 or more, more preferably 60 or more, more preferably 70 or more, preferably 80 or more, more preferably 90 or more, most preferably 100 or more, and preferably 110 or more) Can be mentioned. The protein has a desired biological activity (eg MT1-MMP mediated
Activity that inhibits proMMP-2 activation, activity that inhibits proMMP-2 activation by complex formation between claudin and MT1-MMP, proMMP that complex formation between claudin and proMMP-2 -2 activity that inhibits activation,
Existing in the extracellular domain region of claudin as long as it has an activity of suppressing or inhibiting angiogenesis, cell migration, invasion and / or metastasis resulting from the complex formation It may be a corresponding protein in which a mutation such as substitution, deletion, insertion, transfer or addition of one or more amino acids is appropriately introduced into the amino acid sequence.

As such mutation / conversion / modification methods, chemical methods or claudin family genes (Gen
Bank ^TM / EMBL database (see database), which applies a method commonly used in genetic engineering based on the nucleotide sequence,
For example, edited by The Biochemical Society of Japan, "Sequel Biochemistry Experiment Course 1, Gene Research Method II", p105 (Susumu Hirose), Tokyo Kagaku Dojin (1986);
Japan Society for Biochemistry, "Newborn Chemistry Laboratory 2, Nucleic Acid III (Recombinant DNA Technology)", p233 (Susumu Hirose), Tokyo Kagaku Dojin (199
2); R. Wu, L. Grossman, ed., "Methods in Enzymolog
y ", Vol. 154, p. 350 & p. 367, Academic Press, New
York (1987); R. Wu, L. Grossman, ed., "Methods in
Enzymology ", Vol. 100, p. 457 & p.468, Academic Pr
ess, New York (1983); JA Wells et al., Gene, 3
4: 315, 1985; T. Grundstroem et al., Nucleic Acids
Res., 13: 3305, 1985; J. Taylor et al., Nucleic A
cids Res., 13: 8765, 1985; R. Wu ed., "Methods in E
nzymology ", Vol. 155, p. 568, Academic Press, New
York (1987); AR Oliphant et al., Gene, 44: 17
7, 1986 and the like. For example, site-directed mutagenesis using synthetic oligonucleotides (site-directed mutagenesis) (Zoller et al., Nucl. Acid
s Res., 10: 6487, 1987; Carter et al., Nucl. Acids
Res., 13: 4331, 1986), cassette mutagenesis method (casset
te mutagenesis: Wells et al., Gene, 34: 315, 198
5), restriction site selection mutagenesis
mutagenesis: Wells et al., Philos. Trans. R. Soc.
London Ser A, 317: 415, 1986), Alanine scanning method (Cunningham & Wells, Science, 244: 1081-108).
5, 1989), PCR mutagenesis method, Kunkel method, dNTP [αS] method (Eckstein), region-directed mutagenesis method using sulfite, nitrite and the like.

Further, the obtained protein can be modified in its contained amino acid residue by a chemical method, or an enzyme such as peptidase, for example, pepsin, chymotrypsin, papain, bromelain, endopeptidase, exopeptidase, etc. Can be used for modification or partial decomposition to give a derivative or the like. In some cases, the protein is in vivo or in
It is possible to perform in vitro glucosylation or deglucosylation, and change the position of glucosylation.
(WO87 / 05330; Apin and Wriston, CRC Crit. Rev. Bio
chem., pp.259-306 (1981); Hakimuddin et al., Arch,
Biochem. Biophys., 259: pp.52 (1987); Edge et a
l., Anal. Biochem., 118: pp.131 (1981); "Methods i
n Enzymology ", Vol. 138, pp. 350, Academic Press,
New York (1987) etc.). The protein usually has a C-terminal carboxyl group (-COOH) or carboxylate (-COO).
^- ) But with an amide (-CONH ₂ ) or ester at the C-terminus
It may be (-COOR). Here, R in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl or cyclohexyl, for example, phenyl. , Α-naphthyl, etc.
C _6-12 aryl group, for example, benzyl, phenyl-C _1-2 alkyl group such as phenethyl or α-naphthyl-C _1-2 alkyl group such as α-naphthylmethyl C _7-14 aralkyl group, etc., For example, a pivaloyloxymethyl group commonly used as an oral ester is used. When the protein has a carboxyl group (or carboxylate) other than at the C-terminal, those in which the carboxyl group is amidated or esterified may be included in the desired protein according to the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.

Further, the protein may be a protein having a methionine residue at the N-terminal in the above-mentioned protein, and the amino group of the methionine residue may be a protecting group (for example, C ₁ such as formyl group and acetyl). _-5 protected by a C _1-6 acyl group such as an alkyl-carbonyl group), pyroglutamylated glutamyl group produced by cleavage of the N-terminal side in vivo, on the side chain of an amino acid in the molecule Substituents (eg, —OH, —COOH, amino group, imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (eg, C such as formyl group, acetyl group, etc.)
Those that are protected by _1-6 acyl groups, etc., or complex proteins such as so-called glycoproteins to which sugar chains are bound may be included. Alternatively, it may be expressed as a fusion protein when produced by a gene recombination method, and may be converted or processed in vivo or in vitro into a protein having a biological activity substantially equivalent to that of the desired mutant. . Although a fusion production method commonly used in genetic engineering can be used, such a fusion protein can be purified by affinity chromatography or the like using the fusion portion.

Such fusion proteins include those fused to a histidine tag or those fused to the amino acid sequence of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Can be mentioned. Similarly, the polypeptide can be tagged with a heterogeneous epitope so that it can be isolated and purified by immunoaffinity chromatography using an antibody that specifically binds to the epitope. As typical examples, polyhistidine (poly-His) or polyhistidine-glycine (poly-His-Gly) tag, flu HA tag and its antibody 12CA5, c-myc tag and its antibody 8F9, 3C7, 6E10. , G4, B7 or 9E10, Herpes Sim
plex virus glycoprotein D (gD) tag and its antibody, Flag-peptide, green fluorescent protein (Green Fluo
rescence Protein: GFP), KT3 epitope peptide,
α-tubulin epitope peptide, T7 gene 10 prote
in peptide tag etc. (Field et al., Mo
lecular and Cellular Biology, 8: pp.2159-2165 (198
8); Evan et al., Molecular and Cellular Biology,
5: pp.3610-3616 (1985); Paborsky et al., Protein E
ngineering, 3 (6): pp.547-553 (1990); Hoppet al., B
ioTechnology, 6: pp.1204-1210 (1988); Martin et a
l., Science, 255: pp. 192-194 (1992); Skinner et a
l., J. Biol. Chem., 266: pp.15163-15166 (1991); Lu
tz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,
87: pp. 6393-6397 (1990)). Expression and purification of such a fusion protein can be carried out using a commercially available kit suitable therefor, and can also be carried out according to the protocol disclosed by the kit manufacturer or kit vendor. Modification of protein structure, etc.
For example, referring to "Biotechnology Chemistry Lecture 1, Protein VII, Protein Engineering", edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin (1993), the method described therein or the method described in the literature cited therein, as well as the substance Can be performed in a similar manner. Further, as described below, the biological activity may include immunological activity, for example, having an antigenicity. Among the modifications and alterations, deamination, hydroxylation, phosphorylation, methylation, acetylation, ring opening, ring closure, changing the type of contained sugar chain to a different type, and increasing or decreasing the number of contained sugar chains It may also be a substitution with a D-form amino acid residue. Those methods are known in the art (eg TE Creighton, Prote
ins: Structure and Molecular Properties, pp.79-86
WH Freeman & Co., San Francisco, USA (1983), etc.)
.

Thus, the protein may have one or more amino acid residues that differ from the natural one in terms of identity, or one or more amino acid residue positions that differ from the natural one. The protein has one or more amino acid residues unique to the claudin family of proteins (eg,
1 to 190, preferably 1 to 50, more preferably 1
-20, more preferably 1-10, especially 1-5, etc., lacking deletion analogs, one or more (eg 1-100, preferably 1-50) of the unique amino acid residues. 1 or more (for example, 1 to 100), more preferably 1 to 20, more preferably 1 to 10, especially 1 to 5 etc.) , Preferably 1
50, more preferably 1 to 20, more preferably 1
-10, especially 1-5 etc.) amino acid residues are added. Other than the extracellular domain of claudin, the domain structure or active center structure characteristic of natural claudin is maintained and the resistance to the above-mentioned complex formation is maintained, or via MT-MMPs. All of the above variants are included in the present invention as long as they are resistant to proMMP-2 activation.
In addition, if the protein is other than its extracellular domain,
Those that have a part of the primary structure conformation substantially equivalent to that of the natural claudin may be included, and are resistant to proMMP-2 activation mediated by MT-MMPs. It is believed that those that have biological activity that is substantially the same as that of natural claudin may be included except for some cases. It can also be one of the naturally occurring variants.

The mutein is, for example, human
M1 Δ38-67, M2 Δ55-64, M3 Δ142-155, M4C54S, M5C64S, M6
For ΔYV, M7Δ1-101, those having a homology higher than 50% can be mentioned, and more preferably, 70% to that.
Those having the above or 90% or more homologous amino acid sequences can be mentioned. The fragment derived from the protein is a partial peptide of the protein containing the extracellular domain portion of the claudin (that is, a partial peptide of the claudin protein) and is substantially equivalent to the above-mentioned mutant protein. Any one may be used as long as it has activity. For example, the partial peptide of the protein has an amino acid sequence Asp ^{38 to} Phe ⁶⁷ , Val ^{55 to} Cys ⁶⁴ and As corresponding to the extracellular domain region of human claudin-1.
Among the constituent amino acid sequences of n ^{142 to} Val ¹⁵⁵ , there may be mentioned peptides having at least two consecutive amino acid sequences, preferably 5 or more, more preferably 10 or more, and in some cases 20 or more amino acid sequences.

In the present specification, "substantially equivalent" means the activity of a protein, for example, resistance to proMMP-2 activation mediated by MT1-MMP, proMMP-2 activation inhibitory activity, and corresponding physiological activity. Activity and biological activity are substantially the same. Furthermore, the meaning of the term may include the case of having substantially the same activity, and examples of the substantially same activity include MT1 and MT1 selected from the claudin family. -A property of inhibiting the formation of a complex with MMP, a property of inhibiting the formation of a complex between proMMP-2 and a selected one of claudin family, and a cell associated with activation of proMMP-2 The activity of suppressing and / or inhibiting the migration, invasion and / or metastasis of erythrocytes can be mentioned. The "substantially the same activity" means that the activities are the same in nature, for example, physiologically, pharmacologically or biologically. For example, the activity of inhibiting the complex formation and the activity of inhibiting proMMP-2 activation are equivalent to each other.
(For example, about 0.001 to 1000 times, preferably about 0.01 to 100 times.
Times, more preferably about 0.1 to 20 times, even more preferably about
It is preferably 0.5 to 2 times), but the quantitative factors such as the degree of activity and the molecular weight of the protein may be different.

In some cases, amino acid substitutions, deletions, or insertions often do not result in significant changes in the physiological or chemical properties of the polypeptide, in which case the substitutions, deletions, or The inserted polypeptides will be substantially identical to those without such substitutions, deletions or insertions. Substantially identical substitutions of amino acids in the amino acid sequence can be selected from other amino acids of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, phenylalanine, leucine, isoleucine, valine, proline, tryptophan, methionine, and the like, and polar (neutral) glycine, serine, threonine, cysteine, tyrosine, Examples include asparagine and glutamine, and examples of positively charged amino acids (basic amino acids) include arginine, lysine, and histidine.
Examples of the amino acid having a negative charge (acidic amino acid) include aspartic acid and glutamic acid.

For the synthesis of the protein of the present invention and its partial peptide, a method known in the peptide synthesis field,
For example, chemical synthesis methods such as liquid phase synthesis method and solid phase synthesis method can be used (Stewart et al., Solid-Phase Pept
ide Synthesis, WH Freeman Co., San Francisco, C
A, USA, 1969; Merrifeld, J. Am. Chem. Soc., 85,214.
9-2154, 1963; J. Org. Chem., 37, 3404, 1972; GB
Fields (ed.), "Methods in Enzymology", Vol. 289
(Solid-Phase Peptide Synthesis), AcademicPress, Ne
w York (1997)). In such a method, for example, a resin for protein or peptide synthesis is used, and appropriately protected amino acids are sequentially bound to the desired amino acid sequence on the resin by various condensation methods known per se. For the condensation reaction, various activation reagents known per se are preferably used, and as such a reagent, for example, carbodiimides such as dicyclohexylcarbodiimide can be preferably used. When the product has a protecting group, the desired product can be obtained by appropriately removing the protecting group.
When the protein of the present invention and a part of the peptide thereof are obtained in a free form, they can be converted into a salt by a method known per se or a method analogous thereto, and they are converted into a salt. When obtained, it can be converted into a free form or another salt by a method known per se or a method analogous thereto.

Desired protein (protein fragment having any continuous region in the amino acid sequence present in each claudin or its extracellular domain or a fragment derived therefrom, transmembrane domain of each claudin, cell (Including a protein having an internal domain or a deleted portion or a fragment derived therefrom) is a GenBank ^TM / EMBL
It can be obtained by applying genetic engineering operations based on the nucleotide sequences of mammalian genes such as humans registered in databases such as. For a nucleic acid having a nucleotide sequence of the claudin gene including the human claudin, for example, a DNA molecule, an appropriate primer is designed and synthesized based on the nucleotide sequence of the gene, and preferably the origin of the human claudin nucleotide sequence is used. It can be obtained by PCR amplification or ligase chain reaction (LCR) amplification of a desired sequence using the animal-derived mRNA or cDNA prepared from the mRNA. In some cases, the obtained DNA fragment can be used as a probe to screen a human genomic DNA library or human-derived cDNA library constructed from various human tissues or cultured cells.

A gene encoding a desired polypeptide has a nucleotide sequence to which a start codon, for example, a codon encoding Met (and a stop codon) is added,
In addition, at least the protein encoded by the nucleotide sequence
Has an amino acid sequence with 80% homology and
o Any substance containing a nucleotide sequence having the same effect as that of a peptide having an inhibitory activity against oMMP-2 activation or encoding a peptide having a substantially equivalent biological activity such as an equivalent antigenicity It may be one.
The gene encoding the desired polypeptide is single-stranded DNA
, Double-stranded DNA, RNA, DNA: RNA hybrid, synthetic DNA
Such as human genomic DNA, human genomic DNA library, human tissue / cell-derived cDNA,
It may be any of synthetic DNA. The base sequence of the gene encoding the desired polypeptide may be modified (eg, added, removed, substituted, etc.), and such modified products may be included. Further, as described below, the nucleic acid of the present invention may encode the mutant claudin peptide of the present invention or a part thereof, and preferred examples include DNA. Further, the above-mentioned "equal nucleotide sequence" means, for example, 5 or more consecutive nucleotide sequences of known claudin nucleotide sequences under stringent conditions, preferably 10 or more nucleotide sequences, more preferably 15 nucleotide sequences. Examples include those that hybridize with the above base sequences, more preferably with 20 or more base sequences, and encode an amino acid sequence substantially equivalent to the predetermined one.

Examples of gene recombination techniques that can be used include, for example, J. Sambrook, EF Fritsch & T. Maniatis, "Mol.
ecular Cloning: A Laboratory Manual (2nd editio
n) ", Cold Spring Harbor Laboratory Press, Cold Spr
ing Harbor, New York (1989); DM Glover et al. e
d., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Prac
tical Approach Series), IRL Press, Oxford Universi
ty Press (1995); Biochemical Society of Japan, "Sequence Chemistry Experiment Course 1, Gene Research Method II", Tokyo Kagaku Dojin (1986); Japan Biochemical Society, "Shinsei Chemistry Experiment Course 2, Nucleic Acid III (Recombinant DNA
Technology) ", Tokyo Kagaku Dojin (1992); R. Wu ed.," Methods
in Enzymology ", Vol. 68 (RecombinantDNA), Academi
c Press, New York (1980); R. Wu et al. ed., "Metho
ds in Enzymology ", Vol. 100 (Recombinant DNA, Part
B) & 101 (Recombinant DNA, Part C), Academic Pres
s, New York (1983); R. Wu et al. ed., "Methods in
Enzymology ", Vol. 153 (Recombinant DNA, Part D), 1
54 (Recombinant DNA, PartE) & 155 (Recombinant DN
A, Part F), Academic Press, New York (1987); JH
Miller ed., "Methods in Enzymology", Vol. 204, Aca
demic Press, New York (1991); R. Wu et al. ed., "M
ethods in Enzymology ", Vol. 218, Academic Press, N
ew York (1993) and the like, the methods described in the literature cited therein, or the methods and modifications substantially similar to them (the descriptions in them are referred to in the present specification by referring to them). Included in the disclosure of the document).

First, a sense primer and an antisense primer are synthesized based on the target nucleotide sequence. The primer used in the PCR method is not particularly limited as long as it can amplify a DNA fragment containing a desired site. The sense primer can be preferably selected and synthesized from the 5'end exon site of the gene, and the antisense primer can be preferably selected and synthesized from the 3'end exon site of the gene. More preferably, it can be selected from other than the exon site used for the synthesis of the sense primer. The 5'-end side primer contains at least a start codon or is selected so as to be amplified including the start codon, and the 3'-end side primer contains at least a stop codon, or It is preferable to select such that the stop codon can be included in the amplification.

Although it may be aimed to obtain the full length of the gene cDNA at one time, a plurality of primers are designed and synthesized by utilizing a predetermined exon site (plural exon sites). DNA designed by PCR and cloned based on the DNA fragment thus obtained
The cDNA of the gene can also be obtained from the fragment. For example, using a primer designed based on the base sequence of the exon site on the analyzed 5'end of the exon site of the gene, obtain the 5'end cDNA of the cDNA of the gene, while Analyzed among exon sites of genes
Utilizing a primer designed based on the nucleotide sequence of the exon site on the 3'end side, the cDNA on the 3'end side of the cDNA of the gene is obtained, and then these primers or the obtained Reversed from mRNA isolated from human tissues, especially human brain tissues, using primers designed using the information of the nucleotide sequences of 5'end cDNA and 3'end cDNA of gene cDNA. 1st stra produced by enzyme
The cDNA of the gene can also be obtained by amplification by PCR using the nd cDNA as a template. The 5'-end side primer contains at least a start codon or is selected so as to be amplified including the start codon, and the 3'-end side primer contains at least a stop codon, or It is preferable to select such that the stop codon can be included in the amplification. The primer is preferably an oligonucleotide consisting of 5 or more bases, for example, an oligonucleotide consisting of 10 to 50 bases, more preferably 15 to 35 bases, and even more preferably 18 to 25 bases. The primer can be prepared by a method known in the art, typically by a phosphodiester method, a phosphophotoester method, a phosphoramidite method, or the like.For example, an automatic DNA synthesizer, for example, model 381A DNA synthesizer (Applied Biosystem
s) and the like. The gene can also be obtained by preparing a specific hybridization probe from a predetermined clone, screening a human-derived DNA library, and selecting a clone that hybridizes with the probe. Labeling of the probe and the like with a radioisotope and the like can be carried out using a commercially available labeling kit such as a random primed DNA labeling kit (Boehringer Mannheim). For example, the random-priming kit (Pharmacia LK
B, Uppsala) etc.
A probe having radioactivity can be obtained by labeling with ³² P] dCTP (Amersham) or the like.

As the cDNA library used as the template, commercially available cDNA libraries derived from various tissues can be used directly, and for example, the cDNA libraries commercially available from Stratagene, Invitrogen, Clontech and the like can be used. In a typical example, a gene library prepared from the labeled DNA fragment and human tissues / cells such as human P1 artif
icial chromosome Genomic library (Human Gen
Hybridization is performed using the ome Mapping Resource Center) and a human brain cDNA library (for example, available from Clontech). The human cDNA library is
For example, it can be obtained by constructing into a phage such as λgt10 and infecting it with a host Escherichia coli such as Escherichia coli C600hfl strain to form plaque. If necessary, the cDNA insertion sequence in the clone can be subcloned. The target DNA can also be isolated based on the determined nucleotide sequence. The nucleotide sequence can be determined by the dideoxy method such as M13 dideoxy method.
It can be performed using the Maxam-Gilbert method or the like, but a commercially available sequencing kit, for example, Taq Dye Primer Cycle Sequencing Kit (Applied Biosyste
ms), Sequenase v 2.0 kit or the like, or using an automatic nucleotide sequencer such as a fluorescent DNA sequencer (eg Model 373A, Applied Biosystems). Examples of the polymerase used in the dideoxy method include Klenow fragment of DNA polymerase I, AMV reverse transcriptase, Taq DNA polymerase, T7 DNA polymerase, modified T7 DNA polymerase and the like. A nucleic acid having all or part of the base sequence of the gene can be obtained by chemical synthesis. In that case, the fragments may be chemically synthesized and then they are enzymatically linked. It is also possible to obtain a desired sequence by using a chemically synthesized fragment as a primer or a probe.

In the present specification, the "oligonucleotide" is a relatively short single-stranded or double-stranded polynucleotide, preferably polydeoxynucleotide,
Angew. Chem. Int. Ed. Engl., Vol.28, p.716-734 (19
89), and known methods such as triester method, phosphite method, phosphoamidite method and phosphonate method can be used for chemical synthesis. It is generally known that the synthesis can be conveniently carried out on a modified solid support, for example using an automated synthesizer, which is commercially available. The oligonucleotide may contain one or more modified bases, for example:
It may contain an unnatural base such as inosine or a tritylated base. The obtained PCR product was cloned, the obtained PCR product was identified, and a DN encoding the desired human claudins, MT-MMPs, etc.
You can also get the A fragment. Further, various DNA libraries can be screened in the same manner using this DNA fragment as a probe to isolate the target DNA.
For cloning PCR products, for example, p-Direct (Clon
tech), pCR-Script ^TM SK (+) (Stratagene), pGEM-T (P
romega), pAmp ^™ : Gibco-BRL) and other commercially available plasmid vectors can be used.

In order to obtain a full-length gene sequence containing a complete open reading frame, the DNA fragment obtained as described above is used as a probe, if necessary, and various humans as described above are used. Screening a cDNA library constructed from tissues or cultured cells, selecting a clone that hybridizes to the probe, determining the nucleotide sequence of the cDNA insertion sequence in the clone, and determining the DNA fragment that constitutes the gene. Identify and get. of course,
If necessary, the cDNA insertion sequence in the clone can be subcloned. To isolate the desired DNA, reverse transcription PCR (polymerase chain reaction
led reverse transcription; RT-PCR), RACE (rapid a
mplification of cDNA ends) can be applied. RACE is described, for example, in MA Innis et al. Ed., "PCR P
rotocols "(MA Frohman," a guide to methods and
applications "), pp.28-38, Academic Press, New York
(1990) and the like. The RT-PCR product can be cloned into a plasmid vector and introduced into highly efficient competent cells. Furthermore, a method capable of isolating and purifying mRNA from a trace amount of cells or tissues, for example, REXkit, United States Bioc
hemical; Glass MAX ^TM RNA spin cartridge system, Gi
Using a commercially available kit such as bco-BRL, reverse-transcribe the resulting mRNA using oligo (dT) primer, and
After synthesizing DNA and then attaching a homopolymer tail (eg G residue) to the 3'end of the 1st strand DNA, or attaching an adapter to the DNA, oligo (dT) primer and oligo (dC) primer Alternatively, cDNA can be PCR amplified using adapter primers. A commercially available kit suitable for this is SuperScript ^TM pre-amplifi
cation system (Gibco-BRL); cDNA Cycle ^TM kit (Invi
trogen) and the like.

Hybridization is carried out by constructing a sample (for example, a gene library prepared from human tissues / cells, typically in a phage such as λgt10,
Infect it with host E. coli such as E. coli C600hfl strain,
Plaques obtained by forming plaques, typically plaques formed by microorganisms that retain a given insert DNA, etc.) are transferred to a membrane such as a nylon filter, and if necessary denatured and immobilized. After being subjected to treatment, washing treatment, etc., the substance transferred to the membrane is reacted with a labeled probe DNA fragment modified as necessary in a hybridization buffer. The hybridization treatment is usually performed at about 35 ° C. to about 80 ° C., more preferably about 50 ° C. to about 65 ° C. for about 15 minutes to about 36 hours, more preferably about 1 hour to about 24 hours, The optimum conditions can be selected as appropriate. For example, the hybridization treatment is performed at about 55 ° C. for about 18 hours. The hybridization buffer can be selected and used from those commonly used in the art, for example,
Rapid hybridization buffer (Amersham) etc. can be used. Examples of the denaturing treatment of the transferred film include a method using an alkali denaturing solution, and after the treatment, it is preferable to treat with a neutralizing solution or a buffer solution. The immobilization treatment of the membrane is usually about 40 ° C to about 100 ° C, more preferably about 70 ° C.
It is carried out by baking at about 90 ° C. for about 15 minutes to about 24 hours, more preferably for about 1 hour to about 4 hours, but preferable conditions can be appropriately selected and performed. For example, the immobilization is performed by baking the filter at about 80 ° C for about 2 hours. As the cleaning treatment of the transferred film, a cleaning solution commonly used in this field, for example, 1M NaCl, 1m
This can be performed by washing with 50 mM Tris-HC1 buffer containing M EDTA and 0.1% Sodium Dodecyl sulfate (SDS), pH 8.0. Membranes such as nylon filters can be selected and used from those commonly used in the art, and examples thereof include nylon filters [Hybond-N, Amersham].

The alkali denaturing solution, the neutralizing solution, and the buffer solution can be selected from those commonly used in the art, and examples of the alkali denaturing solution include:
A solution containing 0.5M NaOH and 1.5M NaCl can be mentioned. Examples of the neutralizing solution include 1.5M NaCl.
0.5M Tris-HCl buffer solution, pH 8.0 and the like can be mentioned. Examples of the buffer solution include 2 × SSPE (0.36M NaCl,
20 mM NaH ₂ PO ₄ and 2 mM EDTA) and the like. In addition, prior to the hybridization treatment, it is preferable to pre-hybridize the transferred film, if necessary, in order to prevent a nonspecific hybridization reaction. This prehybridization treatment can be performed, for example, by prehybridization solution [50%
formamide, 5 x Denhardt's solution (0.2% bovine serum albumin, 0.2% polyvinyl pyrrolidone), 5 x SSPE,
Soaked in 0.1% SDS, 100 μg / ml heat-denatured salmon sperm DNA] etc., at about 35 ℃ to about 50 ℃, preferably about 42 ℃, about 4 to about
The reaction can be carried out for 24 hours, preferably for about 6 to about 8 hours, and those skilled in the art can appropriately determine preferable conditions by repeating experiments. Labeled probe used for hybridization
The denaturation of the DNA fragment is performed, for example, at about 70 ° C to about 100 ° C, preferably about 100 ° C, for about 1 minute to about 60 minutes, preferably about 5 ° C.
It can be performed by heating for minutes. The hybridization can be performed by a method known per se or a method analogous thereto, but the stringent conditions in the present specification include, for example, sodium concentration,
About 15 to about 50 mM, preferably about 19 to about 40 mM, more preferably about 19 to about 20 mM, and the temperature is about 35 to about 85 ° C, preferably about 50 to about 70 ° C, more preferably about 60 to about. The conditions of 65 ° C are shown.

After the hybridization is completed, the filter is thoroughly washed to remove the labeled probe other than the DNA fragment of the labeled probe subjected to the specific hybridization reaction. The filter can be washed by using one selected from those commonly used in the art. For example, 0.5 × SSC containing 0.1% SDS (O.15M NaCl,
It can be carried out by washing with a 15 mM citric acid) solution or the like. The hybridized plaque can be typically detected by autoradiography, but can also be appropriately selected from the methods used in the art and used for plaque detection. The plaques corresponding to the detected signal are collected in an appropriate buffer, for example SM solution (100 mM N
50 mM Tris-HCl buffer containing aCl and 10 mM MgSO ₄ , pH 7.
5) etc., and then the phage suspension is appropriately diluted to infect E. coli, and the resulting E. coli is cultured to obtain the target recombinant phage from the cultured E. coli. If necessary, the above-mentioned probe DNA can be used to repeatedly perform the process of screening a target recombinant phage from a gene library or a cDNA library by a hybridization process. The target recombinant phage can be obtained by subjecting cultured Escherichia coli to extraction treatment, centrifugation treatment, or the like.

The obtained phage particles and the like can be purified and separated by a method commonly used in the art, for example, a glycerol gradient ultracentrifugation method (Molecula).
r cloning, a laboratory manual, ed. T. Maniatis, C
old Spring Harbor Laboratory, 2nd ed. 78, 1989) and the like. From phage particles and the like, DNA can be purified and separated by a method commonly used in the art. For example, the obtained phage or the like can be dissolved in a TM solution (10 mM MgSO ₄ containing 50 mM Tris-HCl buffer, pH 7.8). ) Etc., treat with DNase I and RNase A, etc., then
After adding mM EDTA, 50 μg / ml Proteinase K and 0.5% SDS mixed solution, etc. and incubating at about 65 ° C for about 1 hour, this was extracted with phenol and extracted with diethyl ether, followed by ethanol precipitation to precipitate the DNA. The washed DNA was washed with 70% ethanol and dried, and then TE solution (10 mM Trid containing 10 mM EDTA was added.
Obtained by dissolving in s-HC1 buffer, pH 8.0.
Further, the target DNA can be obtained in a large amount by subcloning or the like. For example, the subcloning can be performed using E. coli as a host and using a plasmid vector or the like. The DNA obtained by such subcloning was also subjected to centrifugation,
It can be purified and separated by a method such as phenol extraction or ethanol precipitation.

MT-MMPs-mediated proMMP-2 activators such as claudin family are cloned from mammalian cDNAs such as human-derived cDNA libraries.
Libraries such as various human-derived tissues or cultured cells (in particular, human kidney, brain, pineal gland, posterior pituitary, retina, thymus, testis, ovary, uterus, intestine, heart, liver,
Tissues / cells such as pancreas, small intestine, large intestine, etc.) A plasmid having a cDNA insertion sequence obtained from a cDNA library is activated by at least one of MT-MMPs by a suitable detection system to activate proMMP-2. It is searched by selecting the index that promotes the above. Specifically, for example
Obtained from the cDNA library to be tested, together with a vector expressing the full-length cDNAs of MMP-2 and MT1-MMP
A plasmid having the cDNA insert sequence is transfected into a suitable host cell, for example, latent form, intermediate form, activated MM
This is done by subjecting P-2 to detection and searching for a gene plasmid population that enhances production of activated MMP-2. This search technique can be repeated.

The nucleic acid obtained in the present invention is single-stranded DNA, double-stranded DNA, RNA, DNA: RNA hybrid, synthetic DNA, and the like, and also human genomic DNA, human genomic DNA library, human tissue / cell. It may be derived cDNA or synthetic DNA. The nucleic acid may also hybridize under stringent conditions to those having the characteristic sequences specifically disclosed herein.
The nucleotide sequence of the nucleic acid is modified (eg, added, removed, substituted, etc.) by a method known to those skilled in the art such as gene recombination technology as described above so as to encode a desired protein. , One or more amino acid substitutions or deletions in the amino acid sequence of claudin,
Mutations such as insertion, transfer or addition can be introduced to produce the corresponding protein.

The DNA fragment obtained according to the present invention is transformed into a suitable vector, for example, a plasmid, as described in detail below.
pEX, pMAMneo, pKG5, pET3a (Stratagene) integrated into a vector such as suitable host cells as described in detail below, for example, E. coli, yeast, 293T cells, CHO cells,
It can be expressed in COS cells and the like. Also, the DNA
The fragment is a DN as it is or with an appropriate control sequence added.
It can be introduced as an A fragment or incorporated into an appropriate vector and introduced into cells such as animals. It is also possible to create a transgenic animal that expresses a given gene. Examples of animals include mammals, such as mice, rats, rabbits, guinea pigs, and cows. Preferably, the DNA is used in fertilized eggs of animals such as mice.
The fragments can be introduced to produce transgenic animals. The method can be carried out according to methods known to those skilled in the art, for example, the methods described in US Pat. Nos. 4,736,866 and 4870009.

Confirmation of a predetermined gene product can be carried out using animal cells such as 293T cells, COS-1 cells and the like, which are suitable for it, including tumor-derived cells, etc., which have been transfected with the gene of interest. The foreign gene can be introduced into animal cells such as mammals by a method known in the art or a method substantially similar thereto, for example, calcium phosphate method (for example, FL Graham
et al., Virology, 52: 456, 1973), DEAE-dextran method (eg, D. Warden et al., J. Gen. Viro).
l., 3: 371, 1968, etc.), electroporation method (eg, E. Neumann et al., EMBO J, 1: 841, 1982).
Etc.), microinjection method, ribosome method,
Viral infection method, phage particle method and the like can be mentioned. The gene product produced by the animal cell thus transfected with the predetermined gene can be analyzed. For the analysis, for example, an immunoprecipitation experiment using an antibody such as a monoclonal antibody or Western blotting can be used.

As a plasmid incorporating a predetermined gene, a host cell commonly used in genetic engineering (for example, Escherichia coli,
Prokaryotic cells such as Bacillus subtilis, yeast, 293T cells, CHO cells,
Any plasmid may be used as long as the DNA can be expressed in a eukaryotic cell host such as COS cell and an insect cell host such as Sf21. Within such a sequence, for example, a codon suitable for expression in a selected host cell can be modified, a restriction enzyme site can be provided, and expression of a target gene can be facilitated. Control sequences, promoter sequences, linkers, adapters, etc. that help bind the gene of interest, as well as antibiotic resistance, metabolism control, and detection and selection of reporter sequences. And the like may include useful sequences and the like. Preferably a suitable promoter,
For example, in a plasmid using E. coli as a host, tryptophan promoter (trp), lactose promoter (la
c), tryptophan lactose promoter (tac)
, Lipoprotein promoter (lpp), λ phage P _L
Promoters and the like are SV40 rate promoter, MMTV LTR promoter,
RSV LTR promoter, CMV promoter, SRα promoter, etc.
The GAL10 promoter or the like can be used. Furthermore CYC1, HIS
3, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2,
A control system such as EN0, TP1, AOX1 can also be used.
Enhancers can be inserted into the vector to facilitate transcription of the DNA encoding the desired polypeptide, such enhancers having the effect of acting on a promoter to promote transcription, usually of the order of 10-100 bp. Examples include elements with cis action. Many enhancers are known from mammalian genes such as globin, elastase, albumin, α-fetoprotein and insulin. Typically, an enhancer obtained from a eukaryotic infectious virus can be preferably used, for example, the SV40 enhancer (100-
270 bp), cytomegalovirus early promoter enhancer, polyoma replication origin enhancer in the rate region, and adenovirus enhancer. If necessary, a signal sequence suitable for the host can be added,
Those well known to those skilled in the art can be used.

As a plasmid having E. coli as a host,
For example pBR322, pUC18, pUC19, pUC118, pUC119, pSP64,
pSP65, pTZ-18R / -18U, pTZ-19R / -19U, pGEM-3, pGEM-
4, pGEM-3Z, pGEM-4Z, pGEM-5Zf (-), pBluescript KS
^TM (Stratagene) and the like. Suitable plasmid vectors for expression in E. coli include pAS, pKK22
3 (Pharmacia), pMC1403, pMC931, pKC30, pRSET-B (In
vitrogen) and the like. Examples of plasmids using animal cells as hosts include SV40 vector, polyoma
Examples include virus vectors, vaccinia virus vectors, retrovirus vectors, etc.
D, pcD-SRα, CDM8, pCEV4, pME18S, pBC12BI, pSG5 (S
tratagene) and the like. Yeast-type vector, YEp-type vector, YR
Examples of p-type vector, YCp-type vector, etc.
Examples include pGPD-2. When the host cell is Escherichia coli, examples of the host cell include those derived from the Escherichia coli K12 strain, and examples thereof include NM533, XL1-Blue, C600, DH1, DH5, D.
H11S, DH12S, DH5α, DH10B, HB101, MC1061, JM109, S
TBL2 and the like, and as the B834 strain origin, BL21 (DE3) /
Examples include pLysS. When the host cell is yeast, for example, Saccharomyces cerevisiae, Schizosaccharomyces p
rombe, Pichia pastoris, Kluyveromyces strain, Candida,
Examples include Trichoderma reesia and other yeast strains. When the host cell is an animal cell, for example, African green monkey fibroblast-derived COS-7 cells, COS-1 cells, CV-1 cells, human kidney cell-derived 293 cells, human epidermal cell-derived A431 cells, human colon-derived 205 cells , CO derived from mouse fibroblasts
P cells, MOP cells, WOP cells, Chinese hamster cell-derived CHO cells, CHO DHFR ^- cells, human HeLa cells, mouse cell-derived C127 cells, mouse cell-derived NIH 3T3
Cells, mouse L cells, 9BHK, HL-60, U937, HaK, Jurk
at cells, other transformed cell lines,
Examples include ordinary diploid cells, cell lines derived from in vitro primary culture tissues, and the like. Insect cells include silkworm nuclear polyhedrosis virus (Bombyx mori nuclear polyhe
drosis virus), or a vector derived from it or other appropriate one, and used as a vector for Spodoptera frugiperda (c
aterpillar), Aedes aegypti (mosquito), Aedes albop
ictus (mosquito), Drosophila melangaster (fruitfl
y), silkworm larvae or silkworm cultured cells such as BM-N
Using cells etc. (for example, Luckow e
t al., Bio / Technology, 6, 47-55 (1988); Setlow, J.
K. et al. (Eds), Genetic Engineering, Vol. 8, pp.2
77-279, Plenum Publishing, 1986; Maeda et al., Nat
ure, 315, pp. 592-594 (1985)). Agrobacterium tume
It is also possible to use plant cells as host cells by utilizing faciens and the like, and together with suitable vectors, they are widely known in the art.

In the genetic engineering method of the present invention, in order to modify or convert a restriction enzyme, a reverse transcriptase or a DNA fragment known or used in the art into a structure suitable for cloning. The enzymes such as DNA modifying / degrading enzyme, DNA polymerase, terminal nucleotidyl transferase, and DNA ligase can be used. Examples of restriction enzymes include RJ Roberts, Nuc
leic Acids Res., 13: r165, 1985; S. Linn et al. ed.
Nucleases, p. 109, Cold Spring Harbor Lab., Cold
Spring Harbor, New York, 1982; RJ Roberts, D. M
acelis, Nucleic Acids Res., 19: Suppl. 2077, 1991
And the like. As reverse transcriptase,
For example, the mouse Moloney leukemia virus (mouse Moloney
leukemia virus; MMLV-derived reverse transcriptase (reverse tra
nscriptase), chicken myeloblastosis virus (avian mye
and a reverse transcriptase derived from loblastosis virus (AMV). Reverse transcriptases such as RNase H deficient can be preferably used, and in particular, modified MM lacking RNase H activity.
LV RT can be preferably used, and one having high thermal stability is preferable. Suitable reverse transcriptases include MMLV RT (G
ibco-BRL), Superscript RT plus (Life Technologie
s) etc.

Examples of the DNA polymerase include Escherichia coli
Examples include DNA polymerase, Klenow fragment which is a derivative thereof, Escherichia coli phage T4 DNA polymerase, Escherichia coli phage T7 DNA polymerase, and thermostable bacterium DNA polymerase. Examples of the terminal nucleotidyl transferase include R. Wu et al. Ed., "Methods in Enz
ymology ", Vol. 100, p. 96, Academic Press, New Yor
k (1983) at the 3'-OH end with deoxynucleotides.
Examples include TdTase that adds (dNMP). DNA modification
Examples of the degrading enzyme include exonuclease and endonuclease.For example, snake venom phosphodiesterase, spleen phosphodiesterase, Escherichia coli DNA exonuclease I, Escherichia coli DNA exonuclease III, E. coli DNA exonuclease VII, λ exonuclease, DNase I, nuclease S1. , Micrococcus
coccus) nuclease and the like. Examples of the DNA ligase include Escherichia coli DNA ligase and T4 DNA ligase. DNA Gene cloning and DNA
Suitable vectors for constructing the library include
Examples include plasmids, λ phages, cosmids, P1 phages, F factors, YACs, and the like, and preferably λ phage-derived vectors such as Charon4A, Charon 21A, λg.
t10, λgt11, λDASHII, λFIXII, λEMBL3, λZAPII ^TM
(Stratagene), etc.

The transformant transformed with the expression vector containing the nucleic acid encoding the protein of the present invention stabilizes its high expression ability by carrying out repeated cloning using an appropriate selectable marker if necessary. Can be obtained. For example, in a transformant using an animal cell as a host cell, when the dhfr gene is used as a selection marker, the protein of the present invention is encoded by gradually increasing the MTX concentration and culturing and selecting a resistant strain. It is possible to amplify the DNA and obtain a cell line with higher expression. The transformant of the present invention can be cultured under conditions in which the nucleic acid encoding the protein of the present invention can be expressed to produce and accumulate the desired product. The transformant can be cultured in a medium commonly used in the art. For example, a liquid medium can be preferably used for a transformant using a prokaryotic host such as Escherichia coli or Bacillus subtilis or a yeast host. The medium contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein,
Examples of inorganic or organic substances such as meat extract, malt extract, soybean meal, and potato extract, and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, calcium carbonate, and the like. In addition, yeast, vitamins, casamino acids, growth promoting factors and the like may be added. Further, if necessary, a drug such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. The pH of the medium is about 5-8
Is desirable.

The culture is usually about 15 to about 45 in E. coli.
It is carried out at a temperature of about 3 to about 75 hours, and if necessary, aeration and stirring can be added. When culturing a transformant whose host is an animal cell, as the medium, for example, MEM medium containing about 5 to about 20% fetal bovine serum, PRMI1640 medium, DMEM medium and the like are used. The pH is preferably about 6 to about 8. Culturing is usually performed at about 30 ° C to about 40 ° C for about 15 to about 72 hours, and aeration and agitation are added as necessary. When extracting from the cultured cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and disrupted by ultrasonic waves, lysozyme and / or freeze-thawing. After that, a method of obtaining a crude extract by centrifugation or filtration can be appropriately used. The buffer contains protein denaturants such as urea and guanidine hydrochloride, and Triton X.
A surfactant such as -100 (trade name) or Tween-80 (trade name) may be added. When the desired product is secreted into the culture solution, after the completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.
The target product contained in the thus obtained culture supernatant or the extract can be purified by appropriately combining separation / purification methods known per se, for example, salts such as ammonium sulfate precipitation method. Gel filtration by Sephadex or Sephadex, for example, ion exchange chromatography using a carrier having a diethylaminoethyl group or carboxymethyl group, for example, a carrier having a hydrophobic group such as a butyl group, an octyl group or a phenyl group. It can be obtained by purification by the used hydrophobic chromatography method, dye gel chromatography method, electrophoresis method, dialysis, ultrafiltration method, affinity chromatography method, high performance liquid chromatography method and the like. Preferably, it can be purified and separated by polyacrylamide gel electrophoresis, affinity chromatography in which a ligand such as an antibody that specifically reacts with an antigen such as a monoclonal antibody is immobilized, and the like. For example, gelatin-
Examples include agarose affinity chromatography and heparin-agarose chromatography.

The salt of the protein and a part of the peptide is preferably physiologically acceptable or pharmaceutically acceptable, but is not limited thereto.
Examples of such salts include hydrochloric acid, hydrobromic acid, sulfuric acid,
Nitric acid, salts with inorganic acids such as phosphoric acid, such as acetic acid, formic acid,
Examples thereof include salts with organic acids such as maleic acid, fumaric acid, succinic acid, citric acid, tartaric acid, malic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid and benzenesulfonic acid. Further, examples of the salt include ammonium salts, for example, salts with organic bases such as ethylamine, dimethylamine, trimethylamine and hydroxyethylamine.

The claudin mutants, modified products, derivatives,
The partial peptide fragment and the like can be subjected to the separation / purification treatment as described above. In the present invention, the terms “variant”, “fragment”, “derivative” and “analog” are transcribed from a polypeptide belonging to the claudin family such as the polypeptide of SEQ ID NO: 1 or a base sequence encoding them. Mutated, unspliced or specifically spliced hnRNA or mRNA encoded polypeptide, or in the context of a polypeptide encoded by genomic DNA, a "variant", "fragment", "derivative" thereof. Or “analog”, suppresses or inhibits the biological function or activity of a polypeptide having essentially the same biological function or activity as such polypeptide. A polypeptide having the activity of The polypeptide may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain preferred embodiments, this is a recombinant polypeptide.

When the DNA obtained in the present invention (for example, DNA encoding mutant claudin-1) is transferred to a target animal, it is used as a DNA fragment or the DN.
It is generally advantageous to use A linked downstream of a promoter that can be expressed in animal cells. For example, when the DNA is introduced into a mouse, a gene construct linked to the downstream of various promoters capable of expressing a predetermined DNA derived from an animal having high homology with the mouse in a fertilized egg of a target animal, such as mouse fertilization, is used. By microinjecting into an egg, a transgenic (transgenic) mouse that highly produces mutant claudin-1 can be produced. The mouse is not particularly limited to a pure mouse, for example, C57BL / 6, Balb / C, C3H, (C57BL / 6 x DB
A / 2) F ₁ (BDF ₁ ) and the like. As this promoter, for example, a virus-derived promoter, a ubiquitous expression promoter such as metallothionein, etc. can be preferably used. In addition, when the DNA is introduced, it can be recombined into a recombinant retrovirus and then used. Fertilized mouse eggs into which the DNA of interest is preferably introduced can be grown using, for example, a foster mouse such as ICR. DNA obtained by the present invention at the fertilized egg cell stage (for example, DN encoding mutant claudin-1)
The metastasis of A) is ensured to be present in all germinal cells and somatic cells of the target animal. DNA encoding the polypeptide of interest in embryonic cells of the producing animal after DNA transfer
Is present in all germ cells and somatic cells of the offspring of the producing animal.
Is meant to have. The offspring of this type of animal that has inherited the gene have the potential to express the polypeptide in all of its germ cells and somatic cells.

After confirming that the DNA-introduced animal stably retains the gene by mating, the DNA-introduced animal can be passaged in the usual breeding environment as the DNA-bearing animal. further,
By mating male and female animals carrying the target DNA,
By obtaining a homozygous animal having the transgene on both homologous chromosomes and mating the male and female animals, all the offspring can be passaged so that they have the DNA. An animal into which the DNA has been introduced has high expression of the desired protein, and is therefore useful as an animal for screening the function of the protein. This transgenic animal can also be used as a cell source for tissue culture. For example, by directly analyzing the DNA or RNA in the tissues of transgenic mice, or by analyzing the protein tissues expressed by the gene,
Assays related to proMMP-2 activation via MT1-MMP can be performed. Cells of the tissue containing the polypeptide can be cultured by standard tissue culture techniques and used to study their function, for example, in cells of brain, thymus, testis, brain, intestine, kidney and other tissues. it can. Further, by using the cells, it is possible to contribute to, for example, drug development for enhancing the functions of various tissues. In addition, if there is a highly expressing cell line, the desired polypeptide can be isolated and purified from it. Techniques related to transgenic mice include, for example, Brin
ster, RL, et al.,; Proc. Natl. Acad. Sci. USA,
82: 4438, 1985; Costantini, F. & Jaenisch, R. (ed
s): Genetic manipulation of the early mammalian em
It can be carried out by the method described in the literature such as bryo, Cold Spring Harbor Laboratory, 1985 or the method cited in the literature cited therein, and their modification.

In accordance with the present invention, a coexisting system of one selected from the claudin family, one selected from MT-MMPs (eg MT1-MMP) and proMMP-2 is used,
(i) a substance that inhibits proMMP-2 activation, (ii) a substance that inhibits complex formation consisting of one selected from the claudin family and one selected from MT-MMPs (for example, MT1-MMP) And / or (iii) a screening method for a substance that inhibits the formation of a complex consisting of proMMP-2 selected from the claudin family. For example, MT1-MMP, claudin-1 and proMMP-2 (including a transformant cell expressing the molecule or a culture thereof) can be used to screen substances using proMMP-2 activation as an indicator. . In the screening, for example, MT1-
Using transformant cells expressing MMP, claudin-1 and proMMP-2 or a culture thereof, and between (i) the absence of the test sample and (ii) the contact of the test sample, A comparison will be made regarding the proMMP-2 activation. In addition, in the above screening, the biological activity (eg, protease activity, complex formation, MT1-MMP mediated proMMP mediated
-2 activation, or the resulting angiogenesis, cell migration, invasion and / or metastasis, and physiological and / or biological changes associated with proMMP-2 activation) , Can also be compared.

The expression molecule can be used as it is, but one labeled with a fluorescent substance such as fluorescein, an enzyme or a radioactive substance can also be used. Those having an appropriate tag added by using DNA recombination technology or those having a label added by a chemical method can be preferably used. The activation of proMMP-2 can be measured by a method known in the art, and examples thereof include a method of detecting active MMP-2 with an antibody or the like. Examples of test samples include proteins, peptides,
Examples include non-peptidic compounds, synthetic compounds, fermentation products, plant extracts, tissue extracts of animals and the like, cell extracts and the like. Examples of the test compound used in the test sample preferably include anti-claudin extracellular domain antibody, mutant claudin, claudin extracellular domain peptide fragment and the like, and may particularly include a synthetic compound. Particularly preferred are anti-claudin extracellular domain, pseudo-claudin extracellular domain compound and the like. These compounds may be novel compounds or known compounds. The screening can be carried out according to a measurement method known in the art. Further, it can be carried out by using various labels, buffer systems and other suitable reagents described in the measuring method using the antibody described below, or by following the procedure described there. The measurement is usually carried out in a buffer such as Tris-hydrochloric acid buffer or phosphate buffer that does not adversely affect the reaction, for example, pH 4 to 10
(Preferably, the pH is about 6 to 8).

In the screening of each of these, the measuring system related to the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, textbooks, etc. [eg, Methods in Enzymology, Vol. 1, 2, 5 & 6
(Preparation and Assay of Enzymes); ibid, Vol. 3
(Preparation and Assay of Substrates); ibid, Vol. 4
(Special Techniques for the Enzymologist); ibid,
Vol. 19 (Proteolytic Enzymes); ibid, Vol. 45 (Proteolytic Enzymes)
olytic Enzymes, Part B); ibid, Vol. 80 (Proteolyt
ic Enzymes, Part C) (See above, published by Academic Press (USA)). MT-MMPs with selected from the claudin family using the screen
(In particular, in connection with MT1-MMP mediated activation of proMMP-2, a compound (agonist) or a compound (antagonist) or a salt thereof that promotes the activation of proMMP-2 is screened. Methods are provided, as well as reagents for screening. Compounds or salts thereof obtained using the screening method or screening kit of the present invention, the test compounds described above, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, A compound selected from animal tissue extracts and the like, which promotes or inhibits functions such as activation of proMMP-2 mediated by MT-MMPs (particularly MT1-MMP) by compounds selected from the claudin family Compound. Examples of the salt of the compound include pharmaceutically acceptable salts.

Examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids and the like. Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and aluminum salt and ammonium salt. Suitable examples of salts with organic bases include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N'-
Examples thereof include salts with dibenzylethylenediamine and the like.
Preferable examples of the salt with an inorganic acid include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Suitable examples of salts with organic acids include, for example, formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid,
Examples thereof include salts with citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid and the like.
Suitable examples of salts with basic amino acids include, for example, salts with arginine, lysine, ornithine,
Preferable examples of the salt with acidic amino acid include salts with aspartic acid, glutamic acid and the like.

In the present specification, the term “antibody” is used in a broad sense.
May be used in the sense of the desired claudi
Extracellular domain region of the selected family
And monoclonal antibodies against related peptide fragments
A set of antibodies with specificity for one and various epitopes
A monovalent antibody or a multivalent antibody and
Including polyclonal and monoclonal antibodies
In addition, intact molecules and their fragments
And F (ab '). ₂Fab '
And Fab and include less
Both have two antigen or epitope binding sites.
Chimeric antibody or hybrid antibody, or, for example, mulberry
Dual features such as quadrome and triome
Heterologous recombinant antibody, interspecific hybrid antibody, anti-idiotype antibody
The body, or even if it has been chemically modified or processed
Those considered to be derivatives, known cell fusions or high
Applying bridoma technology or antibody engineering, synthesizing or
Antibodies obtained using semi-synthetic technology, from the perspective of antibody production
Applied the known conventional technology or DNA recombination technology
Antibodies prepared using, as described and defined herein
Neutralizing properties for target antigenic substances or target epitopes
Including antibodies having binding properties or
You can stay. As used herein, the term "antibody" refers to the desired class.
Extracellular domes selected from the Rhodin family
Antibodies as well as antibodies to the in region and related peptide fragments.
The same applies to MMP antibody and anti-MT-MMP antibody.
Be released.

Monoclonal antibodies directed against the antigenic material are produced using any method capable of providing for the production of antibody molecules by a series of cell lines in culture. The modifier "monoclonal" refers to the character of the antibody as being obtained from a population of substantially homogeneous antibodies, which is not considered necessary for the antibody to be produced by any particular method. I won't. Individual monoclonal antibodies, except that there may be small amounts of naturally occurring variants,
It contains a population of antibodies that are identical.
Monoclonal antibodies have a high degree of specificity, which is directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is a single antigen on that antigen. It is aimed at determinants. In addition to its specificity, the monoclonal antibody is also excellent in that it is synthesized by hybridoma culture and is free from or less contaminated with other immunoglobulins. Monoclonal antibodies include hybrid antibodies and recombinant antibodies. They may replace the variable region domain with a constant region domain (e.g., humanized antibody), or replace the light chain with a heavy chain, as long as it exhibits the desired biological activity, regardless of its origin or immunoglobulin class or subclass. It can be obtained by replacement, replacement of one type of chain with another type of chain, or fusion with a heterogeneous protein (eg, US Pat. No. 4,816,567).
Issue; Monoclonal Antibody Production Techniques and
Applications, pp.79-97, Marcel Dekker, Inc., New Y
ork, 1987, etc.). Examples of suitable methods for producing monoclonal antibodies include the hybridoma method (G. Kohler and C.
Milstein, Nature, 256, pp.495-497 (1975)); human B
Cell hybridoma method (Kozbor et al., Immunology To
day, 4, pp.72-79 (1983); Kozbor, J. Immunol., 133,
pp.3001 (1984); Brodeur et al., Monoclonal Antibod
y Production Techniques and Applications, pp.51-6
3, Marcel Dekker, Inc., New York (1987); Trioma method; EBV-hybridoma method (Cole et al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc.,
pp.77-96 (1985)) (Method for producing human monoclonal antibody); U.S. Pat.No. 4946778 (Technology for producing single-chain antibody), and the following references regarding antibodies. :

S. Biocca et al., EMBO J, 9, pp.101-10
8 (1990); RE Bird et al., Science, 242, pp.423-4
26 (1988); MA Boss et al., Nucl. Acids Res., 12,
pp.3791-3806 (1984); J. Bukovsky et al., Hybridom
a, 6, pp.219-228 (1987); M.DAINO et al., Anal. Bio
chem., 166, pp.223-229 (1987); JS Huston et al.,
Proc. Natl. Acad. Sci. USA, 85, pp. 5879-5883 (198
8); PT Jones et al., Nature, 321, pp.522-525 (19
86); JJ Langone et al. (Ed.), "Methods in Enzymol
ogy ", Vol. 121 (Immunochemical Techniques, Part I:
Hybridoma Technology and Monoclonal Antibodies),
Academic Press, New York (1986); S. Morrison et a
L., Proc. Natl. Acad. Sci. USA, 81, pp.6851-6855
(1984); VT Oi et al., BioTechniques, 4, pp.214-2
21 (1986); L. Riechmann et al., Nature, 332, pp.323.
-327 (1988); A. Tramontano et al., Proc. Natl. Aca
d.Sci. USA, 83, pp.6736-6740 (1986); C. Wood et a
l., Nature, 314, pp.446-449 (1985); Nature, 314, p.
p.452-454 (1985) or references cited therein (the descriptions therein are incorporated by reference into the disclosure herein).

Monoclonal antibodies according to the present invention are antibodies of which part of the heavy and / or light chains is derived from a particular species or belongs to a particular antibody class or subclass, so long as they exhibit the desired biological activity. A "chimeric" antibody (immune) that is identical or homologous to the corresponding sequence, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass. Globulin) (US Pat. No. 4,816,567; Morrison et al., Proc. Natl.
Acad. Sci. USA, 81, pp.6851-6855 (1984)). Less than,
The production of the antibody will be described in detail by taking a monoclonal antibody as an example. The monoclonal antibody of the present invention may be a monoclonal antibody obtained by utilizing a cell fusion technique using myeloma cells, and can be produced, for example, by the following steps. 1. Preparation of immunogenic antigen 2. Immunization of animals with immunogenic antigens 3. Preparation of myeloma cells (myeloma cells) 4. Cell fusion of antibody-producing cells and myeloma cells 5. Selection of hybridoma (fused cell) and monocloning 6. Manufacture of monoclonal antibodies

1. Preparation of immunogenic antigen As the antigen, as described above, it is also possible to use an isolated protein fragment of the extracellular domain region of claudins or a fragment derived from it, which can be used for sequencing. Based on the information of the amino acid sequence of the extracellular domain region of claudins (GenBank ^TM / EMBL),
A suitable oligopeptide can be chemically synthesized and used as an antigen. The antigen can be used as it is by mixing with an appropriate adjuvant to immunize an animal, but may be an immunogenic conjugate or the like. The antigen used as an immunogen is the extracellular domain region of claudins (for example, amino acid sequence Asp ^{38 to} Phe of human claudin-1.
⁶⁷ , Val ^{55 to} Cys ^64, and Asn ^{1 42 to} Val ¹⁵⁵ ) may be selected from at least 3 consecutive amino acid residues. For example, a fragment of human claudin-1
Alternatively, it may be a synthetic polypeptide fragment obtained by selecting a characteristic sequence region based on its amino acid sequence, designing a polypeptide, and chemically synthesizing the polypeptide. In addition, the fragment is linked to various carrier proteins through an appropriate condensing agent to give an immunogenic conjugate such as a hapten-protein, which can be used to react with only a specific sequence (or only a specific sequence). Can also be used to design a monoclonal antibody (which can recognize A cysteine residue or the like can be added to the designed polypeptide in advance to facilitate the preparation of the immunogenic conjugate. In binding the carrier proteins, the carrier proteins can first be activated. For such activation, introduction of an activating linking group can be mentioned. As the activated linking group,
(1) Activated ester or activated carboxyl group such as nitrophenyl ester group, pentafluorophenyl ester group, 1-benzotriazole ester group, N-succinimide ester group, etc. (2) Activated dithio group such as 2-pyridyl A dithio group etc. are mentioned. Carrier proteins include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin,
Examples include globulin, polypeptides such as polylysine, and bacterial cell components such as BCG.

2. Immunization of animals with immunogenic antigens can be performed by methods known to those of skill in the art,
For example, Shigeru Muramatsu, et al., Laboratory of Experimental Biology 14, Immunobiology, Maruzen Co., Ltd., 1985, The Biochemical Society of Japan, Sequel Biochemistry Laboratory 5, Immunobiochemistry Research Method, Tokyo Kagaku Dojin, 1986.
, Biochemistry Society, edited by Chemistry Society of Japan 12, Molecular Immunology III, Antigen / Antibody / Complement, Tokyo Kagaku Dojin, 1992, etc. The mammal is immunized with one or more injections of the immunizing agent (optionally with an adjuvant). Typically, the immunizing agent and / or adjuvant is administered to a mammal by multiple subcutaneous injections or intraperitoneal injections. Examples of the immunizing agent include those containing the above-mentioned antigenic peptide or recombinant protein obtained by cloning DNA, the claudin extracellular domain protein of the recombinant protein, or a fragment obtained by enzymatically digesting it. The immunizing agent may be used by forming a conjugate with a protein known to be immunogenic in the mammal to be immunized (for example, the above-mentioned carrier proteins). Examples of the adjuvant include Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG, liposome, aluminum hydroxide, silica, lipid A, synthetic trehalose dicorynomycolate (TDM) adjuvant and the like.
Immunization is performed using, for example, a mouse such as BALB / c, a hamster, and other appropriate animals. The dose of antigen is
For example, about 1 to 400 μg / animal is injected into a mouse, generally intraperitoneally or subcutaneously in a host animal, and thereafter every 1 to 4 weeks, preferably every 1 to 2 weeks, intraperitoneally, subcutaneously or intravenously. Alternatively, booster intramuscularly is repeated about 2 to 10 times. BALB / c mice as well as BALB
It is also possible to use F1 mice of the / c strain mouse and other strain mice. If necessary, an antibody titer measuring system can be prepared and the antibody titer can be measured to confirm the degree of animal immunity. The antibody of the present invention may be obtained from the animal thus obtained and immunized, and includes, for example, antiserum, polyclonal antibody and the like.

3. Preparation of myeloma cells (myeloma cells) The infinitely proliferative cell line (tumor cell cell line) used for cell fusion can be selected from cell lines that do not produce immunoglobulin, such as P3-NS-1-Ag4-1 ( NS-1, Eur. J. Immuno
l., 6: 511-519, 1976), SP-2 / 0-Ag14 (SP-2, Nature,
276: 269-270,1978), P3-X63-Ag8-U1 (P3U1, Curr. Topics Micr) derived from mouse myeloma MOPC-21 cell line
obiol. Immunol., 81: 1-7, 1978), P3-X63-Ag8 (X63,
Nature, 256: 495-497, 1975), P3-X63-Ag8-653 (653,
J. Immunol., 123: 1548-1550, 1979) and the like can be used. 8-Azaguanine-resistant mouse myeloma cell line is added to cell culture medium such as Dulbecco's MEM medium (DMEM medium) and RPMI-1640 medium with antibiotics such as penicillin and amikacin, fetal calf serum (FCS), and
The cells are passaged in a medium supplemented with azaguanine (for example, 5-45 μg / ml), but can be passaged in a normal medium 2 to 5 days before cell fusion to prepare a required number of cell lines. The cell line used was RPMI after thawing the cryopreserved strain completely at about 37 ° C.
The cells may be washed with a normal medium such as I-1640 medium three times or more and then cultured in the normal medium to prepare a required number of cell lines.

4. Cell fusion of antibody-producing cells and myeloma cells 2. The animal, for example, a mouse immunized according to the step of 1. is subjected to spleen extraction 2 to 5 days after the final immunization to obtain a splenocyte suspension. In addition to splenocytes, lymph node cells in various parts of the body can be obtained and used for cell fusion. The splenocyte suspension thus obtained and the above 3. The myeloma cell line obtained according to the step of
(MEM medium), DMEM medium, RPMI-1640 medium or the like, and placed in a cell medium, and a cell fusion agent such as polyethylene glycol is added. As the cell fusion agent, various other ones known in the art can be used.
ting Virus of Japan). Preferably,
For example, 0.5 to 2 ml of 30 to 60% polyethylene glycol
Polyethylene glycol with a molecular weight of 1,000 to 8,000 can be used, and
More preferably, 1,000 to 4,000 polyethylene glycol can be used. The concentration of polyethylene glycol in the fusion medium is preferably 30 to 60%, for example. If necessary, a small amount of dimethyl sulfoxide or the like can be added to promote fusion. The ratio of spleen cells (lymphocytes): myeloma cell line used for fusion may be, for example, 1: 1 to 20: 1, and more preferably 4: 1 to 7: 1. 1 to 10 fusion reactions
Do this for a minute and then add cell culture medium such as RPMI-1640 medium. The fusion reaction treatment can be performed multiple times. After the fusion reaction, the cells are separated by centrifugation or the like and then transferred to a selection medium.

5. Examples of medium for selection of hybridomas (fused cells) and selection for monocloning include FCS-containing MEM medium containing RPMS-1 containing hypoxanthine, aminopterin and thymidine.
Examples of the medium include 640 medium, so-called HAT medium. The selection medium can be replaced generally by adding the same volume as the volume dispensed to the culture plate on the next day, and then replacing it by half with the HAT medium every 1 to 3 days. This can be modified and performed. On the 8th to 16th day after the fusion, the so-called so-called aminopterin was removed.
The medium can be replaced with the HT medium every 1 to 4 days.
Mouse thymocytes, for example, can also be used as a feeder, which may be preferred. The culture supernatant of a culture well in which the proliferation of the hybridoma was grown was subjected to, for example, radioimmunoassay (RIA), enzyme immunoassay (ELISA), fluorescent immunoassay.
(FIA) or other measurement system or fluorescence-induced cell separation device (F
For example, a predetermined fragment peptide is used as an antigen in ACS) or the like, or a target antibody is measured using a labeled anti-mouse antibody to perform screening. The hybridoma producing the desired antibody is cloned. Cloning can be done by picking up colonies in agar or by limiting dilution. The limiting dilution method can be used more preferably. Cloning is preferably performed multiple times.

6. Production of monoclonal antibody The obtained hybridoma strain was FCS-containing MEM medium, RPMI.
The desired monoclonal antibody can be obtained from the culture medium by culturing in an appropriate growth medium such as -1640 medium. In order to obtain a large amount of antibody, ascites of the hybridoma may be mentioned. In this case, each hybridoma is transplanted into the abdominal cavity of a histocompatibility animal of the same strain as the myeloma cell-derived animal and propagated, or, for example, nude
It can be obtained by transplanting each hybridoma to a mouse or the like, allowing it to grow, and collecting the monoclonal antibody produced in the ascites of the animal. Prior to transplantation of hybridoma, the animal can be intraperitoneally administered with mineral oil such as pristane (2,6,10,14-tetramethylpentadecane), and after the treatment, the hybridoma is grown and ascites fluid is collected. You can also The ascites fluid may be used as it is, or may be a conventionally known method, for example, salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography. It can be purified by high performance liquid chromatography or the like and used as a monoclonal antibody. Preferably, the ascitic fluid containing the monoclonal antibody can be purified and separated by ammonium sulfate fractionation, followed by treatment with an anion exchange gel such as DEAE-Sepharose and an affinity column such as a protein A column. Particularly preferably, affinity chromatography in which an antigen or an antigen fragment (eg, synthetic peptide, recombinant antigenic protein or peptide, site specifically recognized by antibody, etc.) is immobilized, affinity chromatography in which protein A is immobilized, hydroxy Examples include apatite chromatography.

Also, transgenic mice or other organisms, such as other mammals, can be used to express antibodies, such as humanized antibodies, to the immunogenic polypeptide products of this invention. It is also possible to determine the sequence of the antibody thus obtained in large quantities, or to prepare the antibody by gene recombination technology using the nucleic acid sequence encoding the antibody obtained from the hybridoma strain. The nucleic acid encoding the monoclonal antibody can be isolated and sequenced by a conventional method such as using an oligonucleotide probe capable of specifically binding to a gene encoding a heavy chain or a light chain of a mouse antibody. . The DNA once isolated can be put into an expression vector as described above and put into a host cell such as CHO or COS. The D
NA can be modified, for example, by substituting a sequence encoding the constant region domain of human heavy chain or light chain in place of the homogenous mouse sequence (Mo.
rrison et al., Proc. Natl. Acad. Sci. USA, 81: 658
1, 1984). Thus, it is possible to prepare a chimeric antibody or a hybrid antibody having a desired binding specificity. Further, the antibody can be modified by preparing a chimeric antibody or a hybrid antibody by applying a chemical protein synthesis technique including the use of a condensing agent as described below. Humanized antibodies can be made by techniques known in the art (eg, Jones et al., Natu
re, 321: pp.522-525 (1986); Riechmann et al., Natu
re, 332: pp.323-327 (1988); Verhoeyen et al., Scie
nce, 239: pp.1534-1536 (1988)). Human monoclonal antibodies can also be produced by techniques known in the art, and human myeloma cells and human / mouse heteromyeloma cells for producing human monoclonal antibodies are known in the art (Kozbor, J. Immunol., 133, pp.30
01 (1984); Brodeur et al., Monoclonal Antibody Pro
duction Techniques and Applications, pp.51-63, Mar
cel Dekker, Inc., New York (1987)). Methods for producing bispecific antibodies are also known in the art
(Millstein et al., Nature, 305: pp.537-539 (198
3); WO93 / 08829; Traunecker et al., EMBO J., 10: pp.
3655-3659 (1991); Suresh et al., "Methods in Enzym
ology ", Vol. 121, pp. 210 (1986)).

Further, these antibodies may be treated with an enzyme such as trypsin, papain, pepsin or the like, and optionally reduced to obtain antibody fragments such as Fab, Fab ′ and F (ab ′) ₂ which may be used. Antibodies can be used in any of the known assays, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniq.
ues, pp.147-158 (CRC Press, Inc., 1987). Any method known in the art can be used to conjugate the antibody to each detectable moiety, for example, David et al., Biochemistry, 13: 1014-1021 (1974); Pain et al, J. Immunol. Meth., 40: pp.219
-231 (1981); and "Methods in Enzymology", Vol. 18
4, pp.138-163 (1990). As the antibody that imparts the labeled substance, an IgG fraction, and further, a specific binding site Fab ′ obtained by digestion with pepsin and reduction can be used. Examples of labeled substances in these cases include enzymes (peroxidase, alkaline phosphatase, β-D-galactosidase, etc.), chemical substances, fluorescent substances, radioactive isotopes, etc. as described below.

The detection / measurement in the present invention can be carried out by immunostaining, for example, tissue or cell staining, immunoassay, for example, competitive immunoassay or non-competitive immunoassay, and radioimmunoassay, ELISA, etc. can be used. The measurement may be performed with or without separation. A radioimmunoassay and an enzyme-immunoassay are preferable, and a sandwich-type assay is also preferable. For example, in a sandwich type assay, one is an antibody against the extracellular domain region and related peptide fragments of those selected from the claudin family of the present invention, the other is selected from the claudin family, MT-MMPs (e.g. MT1MMP etc.) Alternatively, it is an antibody against proMMP-2 and one is detectably labeled. Another antibody capable of recognizing the same antigen is immobilized on the solid phase. Incubation treatment is performed to sequentially react the sample with the labeled antibody and the immobilized antibody, if necessary.
After separating the unbound antibody, the labeled substance is measured. The amount of label measured is proportional to the amount of antigen. In this assay, it is called a simultaneous sandwich type assay, a forward sandwich type assay, a reverse sandwich type assay, etc. depending on the order of addition of the insolubilized antibody and the labeled antibody. For example, washing, stirring, shaking, filtration, pre-extraction of antigen, etc. are appropriately adopted in those measuring steps under specific circumstances. Other measurement conditions such as the concentration of a specific reagent or buffer solution, temperature or incubation treatment time can be changed according to factors such as the concentration of the antigen in the sample and the properties of the sample. Those skilled in the art can appropriately select the optimum conditions effective for each measurement and perform the measurement while using a normal experimental method.

Many carriers are known which are capable of immobilizing antigens or antibodies, and in the present invention, they can be appropriately selected and used. As the carrier, various carriers used for antigen-antibody reaction and the like are known, and in the present invention, it is needless to say that these carriers can be selected and used. Particularly preferably used are, for example, glass, such as activated glass, porous glass, silica gel, silica-
Inorganic materials such as alumina, alumina, magnetized iron, magnetized alloys, polyethylene, polypropylene, polyvinyl chloride,
Polyvinylidene fluoride, polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, crosslinked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, etc. Albumin, collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, natural or modified cellulose such as cellulose acetate, cross-linked dextran, polyamide such as nylon, polyurethane, polyepoxy resin and other organic polymers Substances, those obtained by emulsion polymerization of them, cells, erythrocytes, etc., and if necessary, functional groups such as silane coupling agents. Which are introduced are mentioned. Furthermore, filter paper, beads, inner wall of test container, for example, test tube, titer plate, titer well, glass cell, cell made of synthetic material such as synthetic resin cell, glass rod, rod made of synthetic material, thickening the end, Alternatively, the surface of a solid substance (object) such as a thin rod, a rod having a round protrusion at the end or a flat protrusion, or a thin rod may be used.

An antibody can be bound to these carriers, preferably a monoclonal antibody that specifically reacts with the antigen obtained in the present invention. The binding between the carrier and those involved in these antigen-antibody reactions may be carried out by a physical method such as adsorption, a chemical method using a condensing agent or the like, a chemical method using an activated one, or the like. It can be performed by a method utilizing a chemical bonding reaction. As the label, an enzyme, an enzyme substrate, an enzyme inhibitor, a prosthetic group, a coenzyme, an enzyme precursor, an apoenzyme, a fluorescent substance, a pigment substance, a chemiluminescent compound, a luminescent substance, a chromogenic substance, a magnetic substance, a metal particle such as gold. Examples include colloids and other radioactive substances. As the enzyme, dehydrogenase, reductase, oxidoreductase such as oxidase, for example, amino group, carboxyl group, methyl group, acyl group, transferase that catalyzes transfer of phosphate group, for example, ester bond, Examples thereof include hydrolases that hydrolyze glycoside bonds, ether bonds, peptide bonds and the like, lyases, isomerases and ligases. Enzymes can also be used for detection by using multiple enzymes in combination. For example, enzymatic cycling can be utilized.

Typical labeling isotopes of radioactive substances include [ ³² P], [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C], [ ³⁵ S] and the like. . As a typical enzyme label, peroxidase such as horseradish peroxidase, E. coli β
Examples include galactosidases such as -D-galactosidase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, acetylcholinesterase, catalase, bovine intestinal alkaline phosphatase, and alkaline phosphatase such as Escherichia coli alkaline phosphatase. 4-when using alkaline phosphatase
Fluorescence produced by using umbelliferone derivatives such as methyl umbelliferyl phosphate, phosphorylated phenol derivatives such as nitrophenyl phosphate, enzymatic cycling systems using NADP, luciferin derivatives, and substrates such as dioxetane derivatives, It can be measured by luminescence. The luciferin and luciferase system can also be used. When catalase is used, it reacts with hydrogen peroxide to generate oxygen, and the oxygen can be detected by an electrode or the like. The electrode may be a glass electrode, an ion electrode using a poorly soluble salt film, a liquid film type electrode, a polymer film electrode, or the like. The enzyme label can be replaced with a biotin label and an enzyme-labeled avidin (streptavidin). The label can also use a plurality of different types of labels. In such cases, it may be possible to make multiple measurements continuously, or discontinuously, and simultaneously or separately.

In the present invention, 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine, etc. and horseradish peroxidase are used for signal formation.
Umbelliferyl galactoside, nitrophenyl galactoside and β-D-galactosidase, glucose-6-
A combination of enzyme reagents such as phosphoric acid / dehydrogenase can also be used to form quinol compounds such as hydroquinone, hydroxybenzoquinone and hydroxyanthraquinone, thiol compounds such as lipoic acid and glutathione, phenol derivatives and ferrocene derivatives by the action of enzymes. It is possible to use the one that can be used. Examples of the fluorescent substance or chemiluminescent compound include fluorescein isothiocyanate, for example, rhodamine derivatives such as rhodamine B isothiocyanate and tetramethylrhodamine isothiocyanate, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, acridinium salt, lumiferin, luciferase. , Luminol such as equalin, imidazole, oxalate ester, rare earth chelate compound, coumarin derivative and the like. The labeling can be carried out by utilizing a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, etc. How you can
Furthermore, it can be applied by appropriately selecting from the methods obtained by modifying them. Further, a condensing agent that can be used for preparing the immunogenic complex, a condensing agent that can be used for binding to a carrier, and the like can be used.

Examples of the condensing agent include formaldehyde, glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N, N'-polymethylene bisiodoacetamide, N, N'-ethylene bismaleimide, ethylene glycol bissuccinimine. Dilsuccinate, bisdiazobenzidine, 1-ethyl-3- (3-
Dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (SPD
P), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl ( Four-
Iodoacetyl) aminobenzoate, N-succinimidyl 4- (1-maleimidophenyl) butyrate, N- (ε
-Maleimidocaproyloxy) succinimide (EMC
S), iminothiolane, S-acetyl mercaptosuccinic anhydride, methyl-3- (4'-dithiopyridyl) propionimidate, methyl-4-mercaptobutyrylimidate, methyl-3-mercaptopropionimidate, N- Examples include succinimidyl-S-acetylmercaptoacetate.

According to the assay method of the present invention, a labeled antibody reagent such as a monoclonal antibody in which a substance to be assayed is labeled with an enzyme and the antibody bound to the carrier can be reacted successively or simultaneously. You can also The order of adding the reagents depends on the type of carrier system chosen. When sensitized beads such as plastic are used, a labeled antibody reagent such as a monoclonal antibody labeled with an enzyme is put together with a specimen sample containing a substance to be measured in a suitable test tube, and then the The measurement can be performed by adding beads such as sensitized plastic. In the quantification method of the present invention, an immunological measurement method is used, and as the solid phase carrier in that case, a polystyrene ball, which is well adsorbed to a protein such as an antibody, a polycarbonate ball, a polypropylene ball, or a polypropylene ball,
Various materials and forms such as microplates, sticks, microparticles or test tubes can be arbitrarily selected and used. Optimal pH for measurement, eg pH about 4
It can be carried out in a suitable buffer system so as to keep about 9 to about 9. Particularly suitable buffers are, for example, acetate buffers, citrate buffers, phosphate buffers, Tris buffers, triethanolamine buffers, borate buffers, glycine buffers, carbonate buffers, Tris-HCl. Examples include buffering agents. The buffering agents can be used as a mixture with each other in an arbitrary ratio. Antigen-antibody reaction is about 0 ° C to about 60
Preference is given to working at a temperature between 0 ° C. An antibody reagent such as a monoclonal antibody labeled with an enzyme, an antibody reagent bound to a carrier, and a substance to be measured can be incubated until the equilibrium is reached, but the equilibrium of the antigen-antibody reaction is achieved. The reaction can be stopped after a limited incubation treatment by separating the solid phase from the liquid phase at a much earlier time, and the extent of the presence of a label such as an enzyme in either the liquid phase or the solid phase can be determined. It can be measured. The measurement operation can be performed using an automated measurement device, and the luminescence detector, photo detector, etc. are used to detect and measure the display signal generated by the conversion of the substrate by the action of the enzyme. You can also

In the antigen-antibody reaction, appropriate means can be taken to stabilize the reagents, substances to be measured, labels such as enzymes, and the antigen-antibody reaction itself. In addition, proteins, stabilizers, surfactants, chelating agents, etc. should be added to the incubation solution in order to remove non-specific reactions, reduce inhibitory effects, or activate measurement reactions. It can also be added. Ethylenediaminetetraacetic acid salt (EDTA) is more preferable as the chelating agent. It may be subjected to a blocking treatment which is commonly used in the art or known to those skilled in the art to prevent non-specific binding reaction, for example, normal serum proteins of mammals, albumin, skim milk, milk fermented substances. ,
It can be treated with collagen, gelatin or the like. As long as the purpose is to prevent non-specific binding reaction, those methods can be used without particular limitation. The sample to be measured by the measuring method of the present invention may be a solution or colloidal solution of any form, a non-fluid sample or the like, but preferably a biological sample such as thymus, testis, intestine, kidney, brain, breast cancer. , Ovarian cancer, colorectal cancer, blood, serum, plasma, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, urine, other body fluids, cell culture medium, tissue culture medium, tissue homogenate, biopsy sample, tissue, cells And so on. When applying various analysis / quantification methods including these individual immunological measurement methods to the measurement method of the present invention, it is not necessary to set special conditions, operations and the like. The measurement conditions related to the target substance of the present invention or a substance having substantially the same activity as the target substance of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to the ordinary conditions and operating methods in each method. .

For details of these general technical means, reference can be made to reviews, textbooks, etc. [eg,
Hiroshi Irie, “Radioimmunoassay”, Kodansha, Showa
Published in 1948; edited by Hiroshi Irie, "Sequel Radioimmunoassay"
Kodansha, published in 1979; edited by Eiji Ishikawa et al., “Enzyme Immunoassay”, Medical Shoin, 1983 published by: Eiji Ishikawa et al., “Enzyme Immunoassay” (second edition), Medical Shoin, 1982 Published: Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd edition), Medical Institute, 1987; HV Vunakis et al. (Ed.), "Met
hods in Enzymology ", Vol. 70 (Immunochemical Techn
iques, Part A), Academic Press, New York (1980); J.
J. Langone et al. (Ed.), "Methods in Enzymology",
Vol. 73 (Immunochemical Techniques, Part B), Acade
mic Press, New York (1981); JJ Langone et al.
(ed.), "Methods in Enzymology", Vol. 74 (Immunochem
ical Techniques, Part C), Academic Press, New York
(1981); JJ Langone et al. (Ed.), "Methods in E
nzymology ", Vol. 84 (Immunochemical Techniques, Pa
rt D: Selected Immunoassays), Academic Press, New Y
ork (1982); JJ Langone et al. (ed.), "Methods i
n Enzymology ", Vol. 92 (Immunochemical Techniques,
Part E: Monoclonal Antibodies and General Immunoa
ssay Methods), Academic Press, New York (1983); J.
J. Langoneet al. (Ed.), "Methods in Enzymology",
Vol. 121 (Immunochemical Techniques, Part I: Hybri
doma Technology and Monoclonal Antibodies), Academ
ic Press, New York (1986); JJ Langone et al. (e
d.), "Methods in Enzymology", Vol. 178 (Antibodie
s, Antigens, and Molecular Mimicry), Academic Pres
s, New York (1989); M. Wilchek et al. (ed.), "Meth
ods in Enzymology ", Vol. 184 (Avidin-Biotin Techno
logy), Academic Press, New York (1990); JJ Lango
ne et al. (ed.), "Methods in Enzymology", Vol. 203
(Molecular Design and Modeling: Concepts and Appl
ications, Part B: Anibodies and Antigens, Nucleic
Acids, Polysaccharides, and Drugs), Academic Pres
S., New York (1991), etc., or references cited therein (the descriptions therein are incorporated by reference into the disclosure herein).

Epitope mapping can also be performed using the anti-claudin extracellular domain antibody of the present invention, in particular, a monoclonal antibody. If an antibody that recognizes each epitope is used, a predetermined claudin extracellular domain region and its related peptides can be obtained. It is possible to detect and measure fragments and the like. Antibodies against claudin extracellular domain and its related peptide fragments are associated with proMMPs mediated by MT-MMPs by claudins such as MT1-MMP mediated activation of proMMP-2.
-2 It is useful for the detection and / or measurement of a phenomenon related to activation, and further for the detection and / or measurement of various physiologically active substances caused by excess MMPs activity. The antibody, particularly the monoclonal antibody, (i) detects a disorder, abnormality and / or disease associated with the degradation of tissue or protein by MMPs, or (ii) tumorigenesis of cells caused by the degradation of tissue or protein by MMPs, Angiogenesis, cell migration, invasion, migration and / or metastasis detection or / and (iii) Tumorization of cells, angiogenesis of tumor cells, blood cells, etc., cell migration, invasion , And is useful for detecting migration and / or metastasis or its potential.
It is expected that it can be used to know the degree of motility, invasiveness, chemotaxis and / or metastasis of cancer. In the present invention, claudin induces proMMP via MT-MMPs (eg MT1-MMP).
, A method for detecting and / or measuring a phenomenon associated with claudin-mediated MT-MMPs-mediated proMMP-2 activation, and a method therefor It is possible to provide a reagent, and to detect and / or measure disorders, abnormalities and / or diseases related to proMMP-2 activation via claudin-mediated MT-MMPs,
Detecting tumorigenesis of cells, angiogenesis, migration of cells, invasion, migration and / or metastasis or the possibility thereof, and / or anticancer agent, cancer metastasis inhibitor, angiogenesis inhibitor, Alzheimer's therapeutic agent, joint destruction It can be used as a monitor for determining the effect of therapeutic agents, anti-inflammatory agents and / or immunosuppressive agents. Further, in the present invention, proM via MT1-MMP
A method for detecting and / or measuring a tissue or protein degradation phenomenon due to MP-2 activation and a reagent therefor can be provided. These include controlling the expression of claudin and reagents / compounds therefor.

Furthermore, in the present invention, the extracellular domain region of claudin, for example, the amino acid sequences Asp ^{38 to} Phe ⁶⁷ , Val ^{55 to} Cys ⁶⁴ and Asn ^{142 to} Val ^{155 of} human claudin-1.
A substance having an activity of suppressing or inhibiting the processing of various proteins by MMPs (specifically, MT1-MMP-mediated activation of proMMP-2) is designed by a molecular design based on the one selected from the group consisting of Can be used to get. The substances thus obtained are also within the scope of the idea of the present invention and can be treated as the active ingredient of the present invention. By selecting a particular feature from the sequence and (i) replacing the pharmacophore of that with an isostere,
(ii) replace at least one of the constituent amino acid residues with a D-form amino acid residue, (iii) modify the side chain of the amino acid residue, or (iv) differ from the amino acid residue present in the sequence. This can be done by using techniques adopted in the field, such as arranging and linking amino acid residues, or (v) designing a mimic body by analyzing the three-dimensional structure (for example, Koichi Suto, Pharmaceuticals. Development of Volume 7 (Molecular Design),
Published on June 25, 1990, Hirokawa Shoten Co., Ltd. and references and articles cited therein). Some of these technologies are
It includes the ones described above.

The active ingredient of the present invention is expected to be useful for suppressing or inhibiting the processing of various tissues or proteins by MT-MMPs, for example, the processing of at least proteins such as proMMP-2 by MT1-MMP. Moreover, the active ingredient is useful for suppressing MMPs, for example, MMP-2 activity expression, and is effective in MT1-MMP gene-expressing cells.
It is useful for the prevention or treatment of disorders, abnormalities and / or diseases associated with protein processing by MMPs. In addition, migration, invasion of tumor cells involving MMPs,
Expected to be useful in controlling, eg suppressing, migration and / or metastasis. Claudin variants and their extracellular domain-related peptides are useful for preventing and / or suppressing the migration, invasion and / or metastasis of malignant tumors, ie cancers,
It can be expected as an angiogenesis inhibitor, an antitumor agent and / or a cancer metastasis inhibitor. It is also useful for the prevention or treatment of disorders, abnormalities and / or diseases associated with processing of blood cells by MMPs, and can be expected as an anti-inflammatory agent and / or immunosuppressive agent. Furthermore, it can be expected as a therapeutic agent for Alzheimer's disease, a therapeutic agent for joint destruction and the like. The active ingredient of the present invention can also have a function of enhancing the activation of proMMP-2 by MT1-MMP. The component can also promote a physiological and / or biological response associated with proMMP-2 activation.

The active ingredient of the present invention is the proMMP-2 activation mediated by claudins and MT-MMPs (particularly MT1-MMP), and the proMMP-2 activation, claudins and MT-MMPs.
(Particularly MT1-MMP) complex formation, claudin and pr
There is no particular limitation as long as it has an action of suppressing and / or inhibiting a biological activity such as formation of a complex with oMMP-2, but one having an advantageous action is preferable. The active ingredient is, for example, (a) claudin mutein, a peptide or a salt thereof, a claudin extracellular domain protein, a peptide or a salt thereof, a variant, an analogue or a derivative thereof. etc,
(b) the claudin mutant or a nucleic acid such as DNA encoding the claudin extracellular domain region, (c) the antibody of the present invention, a partial fragment thereof (including a monoclonal antibody) or a derivative thereof, (d) A compound or a salt thereof which suppresses and / or inhibits a biological activity such as a complex formation between claudin and MT1-MMP and a complex formation action between claudin and proMMP-2 is included.

The active ingredient of the present invention includes claudin-related MT-MMPs (particularly MT1-MMP) -mediated processing of proMMP-2, activation of proMMP-2, claudin and MT-MMPs ( In particular, it is expected to be useful for suppressing or inhibiting the process of converting proMMP-2 into an active form by complex formation with MT1-MMP), complex formation with claudin and proMMP-2, and the like. In addition, the active ingredient is useful for maintaining or restoring the function affected and / or lost by excessive activation of proMMP-2, and claudin and MT-MMPs
(Particularly MT1-MMP) complex formation, claudin and pr
o Processing of proMMP-2 caused by complex formation with oMMP-2, eg at least claudin is involved
It is useful for prevention or treatment of disorders, abnormalities and / or diseases associated with activation of proMMP-2. It is also useful for suppressing or inhibiting the processing of proMMP-2 by claudin. In addition, it is useful for controlling, for example, suppressing migration, invasion, and / or metastasis of MT1-MMP gene-expressing cells, and angiogenesis, cell migration associated with processing of proMMP-2 by MT1-MMP in the expressing cells. , Is expected to be useful for the prevention or treatment of disorders, abnormalities and / or diseases caused by infiltration and / or metastasis. Furthermore, active ingredients including claudin extracellular domain proteins of the present invention and antibodies against claudin extracellular domain are useful for controlling angiogenesis, cell migration, invasion and / or metastasis expressing MT1-MMP gene. , Which is useful for suppression, eg pr by complex formation involving the claudin
It is useful for the prevention or treatment of disorders, abnormalities and / or diseases caused by increased angiogenesis, cell migration, invasion and / or metastasis associated with oMMP-2 activation. Particularly, it is useful for preventing and / or suppressing migration, invasion and / or metastasis of tumor cells such as cancer, and can be expected as an antitumor agent and / or a cancer metastasis inhibitor.

Active ingredient of the present invention [eg (a) claudin mutant protein, partial peptide thereof or salts thereof, or analogs, derivatives thereof, extracellular domain peptide fragments of claudin or salts thereof Or a variant, analog, derivative or the like thereof, (b) a nucleic acid such as DNA encoding the variant or extracellular domain peptide fragment, or the like, (c) the antibody of the present invention, or a partial fragment thereof (monoclonal antibody (Including) or a derivative thereof, (d) MT1-MMP
Such as MT-MMPs mediated proMMP-2 activation or a compound or salt thereof that suppresses and / or inhibits biological activity such as complex formation, (e) MT1-MM involving claudin
When an agonist or antagonist of proMMP-2 activation via MT-MMPs such as P or a salt thereof, etc.] is used as a medicine, it is usually used alone or mixed with various pharmaceutically acceptable auxiliary agents to prepare a pharmaceutical composition. Alternatively, it can be administered as a pharmaceutical preparation or the like. Preferably oral administration,
It is administered in the form of a pharmaceutical preparation suitable for topical administration or parenteral administration, and may be in any administration form (including inhalation method and rectal administration) depending on the purpose. In addition, the active ingredient of the present invention may be used in combination with an antitumor agent (anticancer agent), tumor metastasis inhibitor, angiogenesis inhibitor, Alzheimer's therapeutic agent, joint destruction therapeutic agent, anti-inflammatory agent and / or immunosuppressive agent. You can also As an antitumor agent (anticancer agent), tumor transfer inhibitor, angiogenesis inhibitor, Alzheimer's treatment agent, joint destruction treatment agent, anti-inflammatory agent or immunosuppressant agent, any agent that has an advantageous action can be used without limitation,
For example, it can be selected from those known in the art.

The parenteral administration form may include topical, transdermal, intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal administration, but direct administration to the affected area is also possible,
It is also suitable in some cases. Preferably orally in mammals, including humans, or parenterally (eg, intracellular,
Tissue, intravenous, intramuscular, subcutaneous, intracutaneous, intraperitoneal, intrathoracic, spinal cavity, drip, enema, rectal, eardrop, eye drop or nose, tooth, skin or mucous membrane application, etc. ). Specific forms of preparation preparation include solution preparation,
Dispersed preparations, semi-solid preparations, powdered and granulated preparations, molded preparations, leaching preparations and the like can be mentioned, for example, tablets, coated tablets, sugar-coated agents, pills, troches, hard capsules, soft capsules, microcapsules. Capsules, implants, powders, powders, granules, fine granules, injections, solutions, elixirs, emulsions, irrigants, syrups, water solutions, emulsions, suspensions, liniments, lotions , Aerosol, spray,
Inhalants, sprays, ointments, plasters, patches, pasta, poultices, creams, oils, suppositories (for example, rectal suppositories), tinctures, water solutions for skin, eye drops, nasal drops, Powders for ear drops, coatings, infusions, injectables, etc.
Lyophilized preparations, gel preparations and the like can be mentioned.

The pharmaceutical composition can be formulated according to a conventional method. For example, physiologically acceptable carriers, pharmaceutically acceptable carriers, adjuvants, excipients, excipients, diluents, flavors, flavors, sweeteners, vehicles, preservatives, stabilizers, etc. Agent, binder, p
H regulator, buffer, surfactant, base, solvent, filler,
Extenders, solubilizers, solubilizers, tonicity agents, emulsifiers, suspending agents, dispersants, thickeners, gelling agents, curing agents, absorbents,
Adhesives, elastic agents, plasticizers, disintegrants, propellants, preservatives, antioxidants, light-shielding agents, moisturizers, emollients, antistatic agents, soothing agents, etc. are used alone or in combination, and together with this book By admixing the protein of the invention and the like, it can be produced in a unit dose form required for generally accepted formulation practice. Formulations suitable for parenteral use include sterile solutions of the active ingredient and water or other pharmaceutically acceptable medium, or suspensions, such as injections. Generally, water, saline, aqueous dextrose solution, other related sugar solutions, and glycols such as ethanol, propylene glycol, and polyethylene glycol are mentioned as preferred liquid carriers for injection. When preparing an injection, a solution such as distilled water, Ringer's solution, a carrier such as physiological saline, an appropriate dispersant or wetting agent, and a suspending agent can be prepared by a method known in the art. , Suspensions, emulsions, and injectable forms.

Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). A suitable acceptable solubilizing agent, eg alcohol (eg ethanol),
You may use together with polyalcohol (for example, propylene glycol, polyethylene glycol, etc.), a nonionic surfactant (for example, Polysorbate 80 ^™ , HCO-50, etc.) and the like. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.) or reagents for adjusting osmotic pressure, soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human) Serum albumin, polyethylene glycol, etc.), a preservative (eg, benzyl alcohol, phenol, etc.), an antioxidant such as ascorbic acid, an absorption promoter and the like may be added. The adjusted injection solution is usually filled in a suitable ampoule.

For parenteral administration, in a sterile pharmaceutically acceptable liquid such as water, ethanol or oil with or without surfactants and other pharmaceutically acceptable auxiliaries. It is formulated in the form of a solution or suspension. Examples of the oily vehicle or solvent used in the preparation include natural or synthetic or semi-synthetic mono-, di- or triglycerides, natural, semi-synthetic or synthetic fats and oils, such as peanut oil, corn oil, Examples include vegetable oils such as soybean oil and sesame oil. For example, this injection can be usually prepared so as to contain the compound of the present invention in an amount of about 0.1 to 10% by weight. Formulations suitable for topical, eg buccal, or rectal use include, eg, mouth washes, dentifrices, mouth sprays,
Inhalants, ointments, dental fillers, dental coating agents, dental paste agents, suppositories and the like can be mentioned. The mouthwash and other dental agents are prepared by a conventional method using a pharmacologically acceptable carrier. As an oral spray and an inhalant, the compound of the present invention itself or a pharmacologically acceptable inert carrier can be dissolved in a solution for an aerosol or a nebulizer, or can be administered as fine powder for inhalation to teeth and the like. The ointment is prepared by a conventional method by adding a commonly used base such as an ointment base (white petrolatum, paraffin, olive oil, Macrogol 400, Macrogol ointment, etc.).

Medicaments for topical application to the teeth, skin may be formulated in solutions or suspensions of suitably sterile water or non-aqueous vehicles. Additives include, for example, buffering agents such as sodium bisulfite or disodium edetate; preservatives such as acetic acid or phenylmercuric nitrate, benzalkonium chloride or chlorohexidine, and preservatives such as hypromelrose. Examples include thickeners. Suppositories are carriers well known in the art, preferably suitable non-irritating excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc., preferably solid at room temperature but at intestinal temperature. It is prepared by a conventional method using a liquid that melts in the rectum and releases the drug, and is usually prepared so as to contain the compound of the present invention in an amount of about 0.1 to 95% by weight. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Adjuvants such as local anesthetics, preservatives and buffers can be dissolved in the vehicle. Formulations suitable for oral use include solid compositions such as tablets, pills, capsules, powders, granules, and troches, and liquid compositions such as solutions, syrups, and suspensions. Can be mentioned. When preparing the formulation,
A formulation auxiliary agent or the like known in the art is used. Tablets and pills can also be manufactured with enteric coating. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type.

Furthermore, when the nucleic acid such as the DNA of the present invention is used as a therapeutic and / or prophylactic agent as described above, the nucleic acid can be used alone or by a gene recombination technique as described above. It can be used by ligating it to a suitable vector used, for example, a virus-derived vector such as a retrovirus-derived vector. The nucleic acid such as the DNA of the present invention can be administered by an ordinary known method, and as it is, or together with an appropriate auxiliary agent or a physiologically acceptable carrier or the like so that the uptake into cells can be promoted. , Can be formulated and used, and can be administered as a pharmaceutical composition or a pharmaceutical preparation as described above. Also, a method known as gene therapy can be applied. The dose of the active ingredient of the present invention can be selected over a wide range, and the dose and the number of doses can be determined by the sex of the treated patient,
Age and weight, general health condition, diet, administration time, administration method, excretion rate, drug combination, and the degree of medical condition being treated by the patient at that time are determined in consideration of these factors and other factors. .

In manufacturing pharmaceutical products, additives and preparation methods thereof are described in, for example, the Japanese Pharmacopoeia Manual for Editing,
13th Revised Japanese Pharmacopoeia Manual, published on July 10, 1996, Hirokawa Shoten Co., Ltd .; Takashi Ichibanse et al. Development of Pharmaceuticals Volume 12 (Pharmaceutical Formulation [I]), October 1990 Issued on the 15th,
Hirokawa Shoten Co., Ltd .; the same, Pharmaceutical Development Volume 12 (Formulation Material [II]) Issued on October 28, 1990, refer to the description of Hirokawa Shoten Co., Ltd., etc. and select from them as necessary. Can be applied. Kit The present invention is further a pack acceptable in the pharmaceutical field (including analytical / measuring fields such as clinical tests) including one or more containers filled with one or more of the aforementioned composition components of the present invention. And also to the kit. A notice, in the form indicated by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, together with such container (s), for human use A notice (package insert) showing the approval of the relevant government agency regarding the manufacture, use or sale of a product for administration may be attached.

In the present invention, the newly developed expression cloni
Genes associated with the control of proMMP-2 processing via MT1-MMP can be screened by the ng method, especially using 293T cells. Thus proMMP-2 by MT1-MMP
Is one of the genes that accelerate the processing of
The major constituent of ght junctions, claudin-5 (trans
membrene protein deleted in velo-cardio-facial syn
can be identified that encodes the drome [TMVCF]). When Claudin-5 is expressed, by MT1-MMP
Not only does TMP-2 activate proMMP-2 in proMMP-2 activation, but all MT-MMPs mediated proMMP-2
Mutant of MT1-MMP lacking activation and transmembrane domain
It promoted the activation of proMMP-2 via (ΔMT1-MMP). pro
The C-terminal deletion mutant of MMP-2 (proΔMMP-2) is MT in 293T cells.
Processed to an intermediate type by 1-MMP, and then claudin-
It was converted to the active form by the introduction of 5. Pr by MT1-MMP
Compared to the inducing effect of TIMP-2 on oMMP-2 activation, claudi
Activation of proMMP-2 by ΔMT1-MMP in the presence of n-5 and MT1-
The proΔMMP-2 processing by MMPs is dependent on exogenous TIMP-2
On the contrary, the expression was suppressed. These results are
n-5 induces proMMP-2 activation by MT-MMPs
It suggests that TIMP-2 is not involved. MT-MMPs
Enhancement of proMMP-2 activation was also observed in another claudin family, claudin-1, -2, -3. Claudin-
Substitution or deletion of amino acids in the extracellular domain of 1 abolished the promoting effect. Immunoprecipitation analysis showed a direct association between Claudin-1 and MT1-MMP or MMP-2. Not only at the cell-cell boundary but also at other parts of the cell, MT1-MMP
Localized with audin-1. Claudin-1 promoted MT1-MMP-mediated localization of MMP-2 on the cell surface. These results indicate that claudin shows MT-MMP and proM on the cell surface.
Collect MP-2, achieve localized enrichment, resulting in proMMP-2
It is suggested to enhance activation. In the description and drawings, the terms are IUPAC-IUB Commission on Biochemi
cal Nomenclature or based on the meaning of terms commonly used in the art. The meanings of typical terms are shown below. PCR: polymerase chai
n reactionSDS: sodium dodecyl sulfate;

Regarding amino acid sequences of proteins, peptides, etc .: A: alanine residue (Ala) M: methionine residue (Met) C: cysteine residue (Cys) N: asparagine residue (Asn) D: aspartic acid residue Group (Asp) P: Proline residue (Pro) E: Glutamic acid residue (Glu) Q: Glutamine residue (Gln) F: Phenylalanine residue (Phe) R: Arginine residue (Arg) G: Glycine residue ( Gly) S: Serine residue (Ser) H: Histidine residue (His) T: Threonine residue (Thr) I: Isoleucine residue (Ile) V: Valine residue (Val) K: Lysine residue (Lys) W: Tryptophan residue (Trp) L: Leucine residue (Leu) Y: Tyrosine residue (Tyr) Regarding nucleotide sequence: A: Adenine residue G: Guanine residue C: Cytosine residue T: Thymine residue Hind of expression vector pEAK8 described in Example 3 (2) of
III, one of Claudin-1 mutants at EcoRI site M5C64S
Expression vector in which the coding sequence of pEAK was cloned: pEAK
E. coli harboring M5C64S: pEAK M5C64S
1st, 1st, 1st, 1st Higashi, Tsukuba, Ibaraki Prefecture
National Institute of Adva (Postal Code 305-8566)
nced Industrial Science and Technology, Internatio
nal Patent Organism Depositary (IPOD) (deposit number FERM P-18318).

[0105]

EXAMPLES The present invention will be specifically described with reference to examples below, but it is understood that the present invention is not limited to these examples and various embodiments based on the idea of the present specification are possible. It should be. All examples, unless otherwise indicated in detail, are carried out or can be carried out using standard techniques, which are well known and routine to those skilled in the art. .. In the following examples, unless otherwise specified, specific operations and treatment conditions are described in J. Sambrook, E.
F. Fritsch & T. Maniatis, "Molecular Cloning", 2nd
ed., Cold Spring Harbor Laboratory, Cold Spring Ha
rbor, NY (1989) and DM Glover et al. ed.,
"DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practica
l Approach Series), IRLPress, Oxford University Pr
ess (1995); Especially in the PCR method, HA Erliched., PCR
Technology, Stockton Press, 1989; DM Glover et
al. ed., "DNA Cloning", 2nd ed., Vol. 1, (The Prac
tical Approach Series), IRLPress, Oxford Universit
y Press (1995) and MA Innis et al. ed., "PCR
Protocols ", Academic Press, New York (1990), and when using commercially available reagents or kits, attach the instructions (proto) attached to them.
(cols) and attached chemicals are used.

The materials used in the following examples will be described. Dulbecco's modified Eagles's medium (DMEM) was obtained from Nissui. The primers were synthesized by Genset and the human placenta cDNA library was obtained from EdgeBio Systems. Next, MT1-MMP, MT2-MMP, MT3-MMP, MMP-2, MMP-9
And TIMP-2 expression plasmids, as well as monoclonal antibodies against MT1-MMP, MT2-MMP and MT3-MMP are available in the literature: Sat.
o, H., et al., Nature, 370 (6484): 61-65 (1994); T
akino, T., et al., J. Biol. Chem., 270: 23013-23020
(1995); and Tanaka M., et al., FEBS Lett., 402:
219-222 (1997). pro ΔMMP-2
The expression vector is proMMP-having an EcoRI / BglII site.
1st to 1st to 468th amino acids of 2
Obtained by inserting the 1496 DNA fragment into EcoRI / Bgl II of pSG-UT
(GenBank ^TM accession number: NM 004530). pSG-UT is
It has a TGA stop codon after the BamHI site. Also, FLAG-
Tagged MT4-MMP, MT5-MMP and MT6-MMP expression plasmids are described in the literature: Itoh, Y., et al., J. Biol. Che.
m., 274: 34260-34266 (1999); and Kojima, S., et a.
l., FEBS Lett., 480: 142-146 (2000). Polyclonal antibody against Claudin-1 is Zy
Obtained from med. Monoclonal antibody M2 against the FLAG epitope was obtained from Sigma. Claudin-2, Claudin-3
Expression plasmids and polyclonal antibodies of Furus
e, M., et al., J. Cell Biol., 141: 1539-1550 (1998).
And Furuse, M., et al., J. Cell Biol., 147: 891-9.
03 (1999). For cell culture, 293T
Cells and COS-1 cells were cultured in DMEM medium containing 5% fetal bovine serum.

Example 1 Search for proMMP-2 activation regulatory factor via membrane type MMP Plasmid DNA derived from human placenta cDNA library was used as MMP-2,
Human embryonic kidney derived 293T with MMP-9 and MT1-MMP cDNA
Introduced into cells. A human placenta cDNA library constructed on the expression vector pEAK8 (Edge Bio Systems, Gaithersbu
rg, MD) was obtained as an unamplified E. coli clone. 30 bacterial clones were added to 2 ml of MMI medium (4 mM Tris-HCl, p
H 7.2, 1.25% tryptone, 2.5% yeast extract, 125 mM NaC
It was cultured overnight at 37 ° C in 1% and 0.4% glycerol), and the plasmid DNA was extracted by the alkaline SDS method and purified by precipitation with polyethylene glycol. Twenty-four hours before gene transfer, 293T cells were added to DMEM containing 5% fetal bovine serum at 5 x 10 ⁴ cells / well in 96-well (well) microtiter plate.
Mix with 0.1 ml. proMMP-2, proMMP-9, MT1-MMP
An expression vector was prepared by inserting the full-length cDNA of pSG5 (Stratagene) into the cloning site of proMMP-2 expression plasmid 20 ng, proMMP-9 expression plasmid 3 ng, MT1-
30 ng of MMP expression plasmid was introduced with the transfection reagent TransIT LT1 (Mirus, Madiso, WI) together with plasmid DNA (100 ng) prepared from the cDNA library according to its instructions. After culturing at 37 ° C for 48 hours after gene transfer, the medium was removed and the cells were added to 50 microliters of SDS-PAGE sample buffer (50 mM Tris-H
Cl pH6.8, 10% glycerol, 0.1% bromophemol blue,
Solubilized by gentle sonication in 2% SDS).
Cell lysates were processed by gelatin zymography (Kadono, Y.,
et al., Cancer Res., 58: 2240-2244 (1998)). The cell lysate was heated at 37 ° C. for 30 minutes, and 5 microliters was subjected to gelatin zymography.
The results are shown in Figure 1. MMP-2, MMP-9 and MT1-MMP cD
Gene transfer of NA produced a 92 kDa band of latent proMMP-9, a 68 kDa band of latent proMMP-2, and a 64 kDa band of active intermediate MMP-2. It was found that the introduction of the gene of pool cDNA promoted the activation of proMMP-2, resulting in a completely active form (lane 8). proM
Monoclonal cDNA expressing a protein that promotes MP-2 processing was obtained in the second round of screening. proMMP
The cDNA pool that promoted the cleavage of -2 was transformed into E. coli strain XL1-Blue (St
ratagene). 2 for each E. coli colony
It was grown in ml MMI medium and the plasmid DNA was purified. MT1
-For DNA clones that promoted activation of proMMP-2 by MMP, LI-COR DNA sequencer Model4200L (S) -2
Was used to determine the inserted nucleotide sequence. 1.4kb cDNA
The nucleic acid sequence of the fragment was determined. This cDNA clone was identified as velo-cardio-facial syndr by homology search of nucleic acid sequences.
ome (TMVCF) (GenBank ^TM accession number: NM 00327
7; Morita, K., J. Cell. Biol., 147: 185-194 (199
It was shown in 9)) that the deletion was derived from the human gene for claudin-5 / transmembrane protein.

Example 2 (1) Claudin-5 of proMMP-2 processing by MT-MMPs
(A) Claudin- in activation of proMMP-2 by MT1-MMP
The effect of 5 expression was tested using 293T cells. 293T cells were seeded in 24-well microwells 24 hours before gene transfer. Control plasmid (pSG5) or claudin-
5 plasmids (50 ng) to MMP-2 plasmid (80 ng) and each MT-MMPs plasmid (220 ng) shown in Figure 2A.
At the same time, the cogene was introduced into 293T cells cultured in a 24-well culture plate. Total plasmid DNA with pSG5 plasmid
It was adjusted to 500 ng / well. 48 hours after gene transfer, cells were collected, solubilized with 150 microliters of SDS-PAGE sample buffer, and after heating at 37 ° C for 30 minutes, 15 microliters of cell lysate was treated as in Example 1. And analyzed by gelatin zymography. The results are shown in Figure 2A.
Expression of Claudin-5 alone did not affect proMMP-2 processing. For transfection of MT1-MMP or MT3-MMP cDNA, use proMMP-
2 was converted from the latent form to the activated intermediate. Introduction of claudin-5 cDNA into these cells promoted processing to produce the fully active form. MT2-MMP, MT4-MMP, MT5-
Expression of MMPs and MT6-MMPs did not induce apparent proMMP-2 processing, but claudin-5 cDNA was expressed in these MTs.
-When transfected with MMPs, proMMP-2 activation was observed. The cell lysate used in the gelatin zymography described above was subjected to SDS-PAGE under reducing conditions, and the expression of MT-MMPs was examined by Western blotting using each monoclonal antibody. The results are shown in Figure 2B. In Figure 2B, all cells were co-transfected with MMP-2 and a plasmid as indicated. In addition, MT1-MMP and TIMP-2 cDNA were co-transfected. It was confirmed by immunoblot analysis that the expression of MT-MMPs was not affected by the expression of claudin-5. Expression of TIMP-2 induced the accumulation of 57-kDa MT1-MMP, but expression of claudin-5 did not (Fig. 2B, upper left).

(B) Acceleration of proMMP-2 processing by MT1-MMP mutant by Claudin-5 Control plasmid (pSG5) or claudin-5 plasmid (40 ng) was added to MMP-2 plasmid (80 ng) and wild type MT1- The cogene was introduced into 293T cells cultured in a 24-well culture plate together with MMP (WT) or its mutant plasmid (220 ng) as shown in FIG. Total plasmid DNA
Was adjusted to 500 ng / well with pSG5 plasmid. Forty-eight hours after gene transfer, cells were harvested and analyzed by gelatin zymography. The results are shown in Figure 3A. In addition, the cell lysate used for the gelatin zymography described above was subjected to reducing conditions.
Subjected to SDS-PAGE, MT1-MMP mutant Δ535 (Ala ^{536 and} later deleted, SWISS PROT accession No. P50281) or Δ335 (P
After deletion of he ^336, the expression of SWISS PROT accession No. P50281) was examined by Western blotting using a monoclonal antibody against MT1-MMP. The results are shown in Figure 3B. MT
The transmembrane domain of 1-MMP is important for cell-associated proMMP-2 activation, and MT1-MMP lacking the transmembrane domain (Δ535) or MT1-MMP lacking the transmembrane and fibronectin-like domains (Δ335) Showed only a faint proMMP-2 activation function, but Claudin-5 and each MT1-MMP
Co-expression with the mutant induced proMMP-2 activation (Fig. 3A). It was confirmed by immunoblot analysis that the expression of MT1-MMP mutant was not affected by the expression of claudin-5 (Fig. 3B).

(2) TIMP-2 independent proMMP-2 activation In one model of proMMP-2 processing, MT1-MMP,
It is involved in the formation of a trimolecular complex composed of TIMP-2 and proMMP-2. Therefore, proΔMMP-2, which lacks the C-terminal domain for interacting with TIMP-2, is MT1-MMP.
It is believed to be insensitive to processing by. Therefore, claudi for proΔMMP-2 processing
The effect of n-5 expression was tested on 293T and COS-1 cells. pr
o ΔMMP-2 plasmid (40 ng) as control plasmid (pSG5), MT1-MMP plasmid (220 ng) and claud
293T cells cultured in 24-well culture plates were co-transfected with the in-5 plasmid (50 ng). All plasmids
DNA was adjusted to 500 ng / well with pSG5 plasmid. Forty-eight hours after gene transfer, cells were harvested and analyzed by gelatin zymography. ProΔMMP-2 expressed in 293T cells
Was detected as a 48 kDa band in gelatin zymography (Fig. 4). Interestingly, co-expression of MT1-MMP in 293T cells resulted in 44 kD of proΔMMP-2.
a Processed to intermediate. Introduction of Claudin-5 cDNA promoted MT1-MMP-mediated processing of proΔMMP-2, resulting in a 42 kDa active form. proΔMMP-2 and clau
Processing did not occur with gene transfer of din-5 cDNA alone.

In order to investigate the relationship between proΔMMP-2 processing by MT1-MMP and TIMP-2, the effect of TIMP-2 expression on the processing by MT1-MMP was investigated using proMMP-2 and proΔ.
Comparison was made between MMP-2. The results are shown in Figures 5A and 5B. proM
MP-2 (80 ng) or proΔMMP-2 (40 ng) plasmid and MT1-MMP (220 ng) were added to TIMP in the amounts shown in FIG.
-Two plasmids were cultured in 24-well culture plates 29
The cogene was introduced into 3T cells (5A) or COS-1 cells (5B).
Forty-eight hours after gene transfer, cells were harvested and analyzed by gelatin zymography. The total plasmid DNA was adjusted to 500 ng / well with pSG5 plasmid. In 293T cells (Fig. 5A), proMMP-2 is processed by MT1-MMP into an intermediate. When TIMP-2 was expressed, it activated proMMP-2 activation in a dose-dependent manner and produced an active form. In contrast, MT1-MMP processing of pro ΔMMP-2 is associated with TIMP-2.
The expression suppressed proMMP-2 activation in a dose-dependent manner. CO
In S-1 cells (Fig. 5B), proMMP-2 is exogenous TIMP-2.
Converted to the fully active form by MT1-MMP without expression of
There was no further induction. However, high doses of TIMP-2c
It was disturbed by transfection of DNA. proΔMMP-2
Only 44kDa and 42kD without expression of exogenous TIMP-2
Only a body was generated, which was only inhibited by the expression of TIMP-2.

(3) TIMP-2 enzyme-linked immunosorbent assay (ELIS
A) 293T cells were placed in a 24-well microwell with an expression vector
After culturing for a period of time, replace the culture medium with 0.5 ml FCS-free medium,
It was further cultured for 16 hours. COS-1 cells were treated similarly. The TIMP-2 concentration in the culture medium was (Fujimoto, N., Zhan
g, J., Iwata, K., Shinya, T., Okada, Y., and Hayak
awa, T. (1993) Clin Chim Acta 220, 31-45.) ELI
Measured by SA (n = 3). TIMP-2 levels secreted by 293T and COS-1 cells were compared with and without exogenous TIMP-2 expression (Table 1).

[0113]

[Table 1]

Endogenous TIMP-2 levels were below the limit of detection in 293T cells (<1 ng / ml). Endogenous TIMP-2 levels (49 ng / ml) in COS-1 cells were similar to those that induced maximal proMMP-2 activation in 293T cells (10
1 ng / ml). ProMMP-2 activation by MT1-MMP is synthesized by TIMP-2 in COS-1 cells within the range of 49-1630 ng / ml.
It was almost constant under the conditions of expression. The range was also similar in 293T cells (101-995 ng).
/ ml). Complete inhibition of proMMP-2 activation by MT1-MMP was observed when TIMP-2 levels reached 7280 ng / ml in medium.

The effect of TIMP-2 on proMMP-2 activation mediated by the MT1-MMP mutant Δ535 was investigated. The results are shown in Figure 5C. Control plasmid or claudin-5 plasmid (50 ng), proMMP-2 (80 ng) and MT1-MMP mutant Δ535 (220 ng) in 24-well culture plates with the amount of TIMP-2 plasmid shown in Figure 5C. Genes were introduced into the cells cultured in 1. Total plasmid DNA with pSG5 plasmid
It was adjusted to 500 ng / well. Forty-eight hours after gene transfer, cells were harvested and analyzed by gelatin zymography. Δ53
Expression of 5 alone did not effectively induce proMMP-2 processing. Also, introduction of TIMP-2 plasmid at various amounts did not show the effect as observed with wild type MT1-MMP. Activation of proMMP-2 by Δ535 is claudin-
It was observed only when 5 was co-expressed, and TIMP was dose-dependently observed.
-2 was inhibited by expression. proMMP with MT2-, MT4-, MT5- and MT6-MMP detected only in the presence of claudin-5
-2 activation (Fig. 2A) was not promoted, but not inhibited by TIMP-2 expression. These results show that TIMP-2
It is suggested that it is not related to proMMP-2 activation mediated by claudin-5.

Example 3 (1) Claudin- of MT-MMP-mediated proMMP-2 processing
Claudin-1,-, another member of the Claudin family promoted by 1, -2 and -3
2 and -3 were used to examine the promotion of proMMP-2 processing via MT1-MMP. MMP-2 (80ng) and MT1-MMP (220n
g) The plasmid was co-transfected with 293T cells cultured in a 24-well culture plate together with the control or the claudin plasmid (50 ng) shown in the upper panel of FIG. Total plasmid DNA was combined with pSG5 plasmid at 500 ng / well. At 48 hours after gene transfer, cells were collected and analyzed by gelatin zymography (FIG. 6, upper panel). The cell lysate was also subjected to SDS-PAGE under reducing conditions, and the presence of claudin was examined by Western blotting using the antibodies shown in the lower panel of FIG. 6 (lower panel of FIG. 6).
Co-expression of MT1-MMP with each claudin enhances proMMP-2 processing, 62kD similar to that observed with claudin-5.
a generated active form.

(2) Cloning of Claudin-1 cDNA and Preparation of Mutant Using human placenta total RNA with an 8 mer random primer and ReverTra Ace reverse transcriptase (TOYOBO, Japan)
cDNA was synthesized. Claudin- using the following primers
1 cDNA (GenBank ^™ accession number AF134160) was obtained. Incidentally, the amino acid sequence of human Claudin-1 is, MANAGLQLLG FILAFLGWIG AIVSTALPQW RIYSYAGDNI VTAQAMYEGL WMSCVSQSTG QIQCKVFDSL LNLSSTLQAT RALMVVGILL GVIAIFVATV GMKCMKCLED DEVQKMRMAV IGGAIFLLAG LAILVATAWY GNRIVQEFYD PMTPVNARYE FGQALFTGWA AASLCLLGGA LLCCSCPRKT TSYPTPRPYP KPAPSSGKDY V [sequence listing SEQ ID NO: 1] is.

EcoRI site starting from number 202 (underlined)
Added positive primer: 5'Cl-1

[Chemical 1] [SEQ ID NO: 2]

EcoRI site starting from 886th place (underlined)
Reverse primer added: 3'Cl-1

[Chemical 2] [SEQ ID NO: 3] The amplified cDNA fragment is the expression vector pE
It was cloned at the EcoRI site of AK8 (pEAK-MI). pEAK-M
The vector I is the antisense oligomer AAG CTT GAA TTC GGC GCG CCA GAT ATC GCA TGC [SEQ ID NO: 4] sense oligomer CGC CGG CGC CAT GGC GAT CGG CTA GCA GAT CT [SEQ ID NO: 5]. PEAK8 cut with I
Created by inserting into.

[0120] A series of Claudin-1 mutants was prepared by two-step PCR using the above-mentioned wild-type Claudin-1 as a template.
The first step is antisense, sense primer and 5'Cl-1 or 3'Cl containing a mutation specific to the wild type sequence.
-1 consisting of 2 reactions with primers. According to this, 5'Cl-1 and the appropriate antisense primer amplifies the upstream part of the fragment containing the mutation, and 3'Cl-1 and the corresponding sense primer amplifies the downstream part. Mix the products 5'Cl-1
And 3'Cl-1 were used as the only primers for the second PCR. Full-length constructs are amplified only by annealing the upstream and downstream halves in which the mutation was introduced. The fragment amplified by PCR was cut with EcoRI and cloned with pEAK-MI. Mutants were created with the following set of mutation primers.

MlΔ38-67, antisense CAA GGA GTC GCC GGC ATA GGA GTA [SEQ ID NO: 6] sense GCC GGC GAC TCC TTG CTG AAT CTG [SEQ ID NO: 7] M2Δ55-64, antisense GAC TTT GCA GGA CAT CCA CAG CC [SEQ ID NO: 8] sense ATG TCC TGC AAA GTC TTT GAC TC [SEQ ID NO: 9] M3Δ142-155, antisense CCT GGC ATT GCC ATA CCA TGC TGT G [SEQ ID NO: 10] sense TAT GGC AAT GCC AGG TAC GAA TTT G [SEQ ID NO: 11]

M4C54S, antisense CTC TGC GAC ACG GAG GAC ATC [SEQ ID NO: 12] sense CTG TGG ATG TCC TCC GTG TCG [SEQ ID NO: 13] M5C64S, antisense TCA AAG ACT TTG GAC TGG ATC [SEQ ID NO: 14] ] Sense GGC AGA TCC AGT CCA AAG TCT [SEQ ID NO: 15] The M6ΔYV mutant in which the PDZ motif on the C-terminal side was destroyed was prepared by PCR using the following primer set. Antisense CCG AAT TCT GCA CCT GCC ACC [SEQ ID NO: 16] Sense GAC GGA TTC CAC TTA GTC TTT CC [SEQ ID NO: 17]

The obtained PCR product was the expression vector Hind.
III, cloned at EcoRI site. The M7Δ1-101 mutant lacking the N-terminal half was prepared by PCR using the following primer set. Antisense CAA GCT TGG CAT GAA GTG TAT GA [SEQ ID NO: 18] Sense TGA ATT CGA CAG ATC ACA CGT AG [SEQ ID NO: 19] The obtained PCR product was cloned at the Hind III and XbaI sites of the expression vector. . FIG. 8 shows a schematic diagram of the gene structure of claudin-1 and its mutant.

(3) Disappearance of promoting effect by mutation of Claudin-1 extracellular domain A defect and an amino acid substitution were introduced into Claudin-1. These mutants were examined for their effect of promoting proMMP-2 processing mediated by wild type MT1-MMP or MT1-MMP mutant Δ335. The result is shown in FIG. In Figure 7A, MT
1-MMP (upper panel) or Δ335 (lower panel) plasmid and MMP-2 plasmid were used as control plasmid (lane C), claudin-1 (lane W) or the mutant plasmid shown in FIG. 7 (of the gene structure of the mutant). A schematic diagram is shown in FIG. 8), and 293T cultured cells were transduced in a 24-well culture plate. Cells were harvested 48 hours after transduction and analyzed by gelatin zymography. Cell lysate
It was also subjected to SDS-PAGE under reducing conditions, and claudin-1 was used for clautin by Western blotting.
The presence of the din-1 mutant was confirmed (Fig. 7B). Amino acid residues 38-67 or 55 in the first extracellular domain of Claudin-1
Deletion of ~ 64 is caused by proM mediated by MT1-MMP or Δ335 mutant
The stimulatory effect on MP-2 activation was abolished. Clau lacking amino acid residues 142-155 in the second extracellular domain
The din-1 mutant clearly reduced the promoting effect. However, low levels of activity remained. Substituting cysteine 54 or cysteine 64 in the first extracellular domain of Claudin-1 with serine greatly reduced the enhancing effect. Deletion of C-terminal tyrosine and valine (PDZ domain) had no clear effect. Deletion of N-terminal 101 amino acid residues completely abolished the promoting effect of claudin-1. These results show that the extracellular domain, especially the first extracellular domain,
It is suggested that it is important in promoting proMMP-2 activation via MT-MMPs.

(4) Claudin-1 and MT1-MMP by immunoprecipitation
The interaction between Claudin-1 and MT1-MMP was investigated by immunoprecipitation. Incubated on a 60 mm diameter plastic plate
TransIT LT-1 was used in 293T cells and MT1-MMP, MMP-2 or claudin-1 plasmids adjusted to 2 microgram each were introduced, and transient expression was carried out for 24 hours, and then 50 microcurie / ml. [S-35] Methionine and Cysteine (Ame
(rsham Pharmacia Biotech) for 4 hours. Cells should be in cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM N
aCl, 1% Triton X-100 (v / v), 0.5% sodium deoxychola
te, 0.1% SDS, 1 mM phenylmethyl sulfonylfluoride)
, And centrifuged at 15,000 rpm for 10 minutes to collect the supernatant. One microgram of anti-claudin-1 polyclonal antibody or anti-MT1-MMP or anti-MMP-2 monoclonal antibody was added to the supernatant and incubated overnight at 4 ° C. Antigen-
Antibody conjugates are loaded with Protein G Sepharose beads (Amersham
Pharmacia Biotech) and the beads were washed 3 times with cell lysis buffer. Material bound to beads is 1% 2-mer
The sample was eluted with SDS-PAGE sample buffer containing captoethanol, boiled for 2 minutes, and then separated on a 12% polyacrylamide gel. The substance labeled with [S-35] was detected with a bioimage analyzer (Fuji, Tokyo, Japan).

The results are shown in FIG. A polyclonal antibody against Claudin-1 precipitated a 22 kDa claudin-1 protein from 293T cells transduced with claudin-1 cDNA. MT1-MMP
In 293T cells in which both claudin-1 and claudin-1 were introduced, cl
MT1-MMP sedimented with audin-1. But MT1-MMP
MT1-MMP was not precipitated when only cDNA was introduced. Monoclonal antibodies against MT1-MMP
Both 60-kDa band and MT1-MMP and claudin-1 cDNA were precipitated from cells co-transfected with MT1-MMP and claudin-1 cDNA, but clautin-1 cDNA alone was clautin-1.
din-1 was not allowed to settle. Claudin-1 is MMP-2
Antibodies to MMP-2 co-precipitated from cells into which both MMP-2 and claudin-1 cDNA had been introduced, but not from cells into which each cDNA had been introduced.

Example 4 (1) Claudin-5 to which FLAG or Green Fluorescence Protein (GFP) was added was constructed. Multiple cloning site and FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-L
The ys) coding region was cloned into the pFLAG-CTC plasmid DNA (I
BI FLAG Biosystems, USA) as a template, N26 and C24 (IBI F
It was amplified by PCR using LAG Biosystems, USA) as a primer. This was cleaved with Hind III and Bgl II and then inserted into the cloning site of pSG5 (referred to as pSG-FLAG). Claudin-5 expression plasmid with FLAG epitope
Din-5 was constructed for detection with anti-FLAG antibody M2. Claudin-5 with the stop codon replaced by the restriction enzyme Bgl II site
The cDNA consists of T7 positive primer and 774th position (GenBank ^TM Accessi
on No. NM 003277) Bgl II site (underlined)
It was amplified by PCR using the reverse primer with.

[Chemical 3] [SEQ ID NO: 20] The amplified fragment was cleaved with EcoRI and BglII and inserted into the EcoRI and BglII sites of the pSG-FLAG vector (referred to as pSG-Claudin-5-FLAG).

(2) A Claudin-5-GFP chimeric protein was prepared to monitor the localization of Claudin-5 with a confocal laser scanning microscope. The region encoding GFP is the positive primer in which the ATG of the start codon is replaced with the Bgl II site.
It was amplified by PCR with a reverse primer starting downstream of the stop codon to which the BglII site was added. The amplified fragment is Bgl
Cleaved-of pSG-Claudin-5-FLAG plasmid cleaved with II
5 It was inserted into the Bgl II site between the cDNA fragment and the FLAG region (designated pSG-Claudin-5-GFP). GFP-tagg was added to 293T cells cultured in a 0.2 mm thick glass bottom 35 mm diameter plate.
ed MT1-MMP (MT1-GFP) plasmid (1 μg) was transduced,
MT1-MMP plasmid (1 μg) or claudin-5 tagged w
The ith GFP (Claudin-5-GFP) plasmid (1 μg) was introduced. Fluorescence was observed using a confocal laser microscope. MT
MT1-GFP in cells transfected with 1-GFP plasmid only
Was detected on the cell surface and cytoplasmic granules. Claudin-5-
GFP was found at cell boundaries and cytoplasmic granules. Its localization was not significantly changed by the expression of MT1-MMP. MT
The effect of claudin-1 expression on the cell surface localization of 1-MMP was measured using MT1-GFP plasmid (1 μg) and claudin-1 plasmid (1 μg).
μg) for expression. The transduced cells were immunostained with anti-claudin-1 antibody and goat anti-rabbit IgG antibody conjugated with TRITC. It was observed with a confocal laser microscope. It was shown that MT1-GFP co-localizes with claudin-1. Claudin-1 was localized not only at the cell border but also in the cytoplasm. MT1-GFP and claudin-1 at cell boundaries
Co-localization of was clearly demonstrated in cells into which both cDNAs were introduced. Furthermore, part of MT1-GFP also colocalized with claudin-1 in other parts of the cell.

(3) Colocalization of MMP-2 and MT1-GFP The cDNA fragment of MT1-MMP was cloned into pCEP4 (Invitorgen). Mutations were introduced into the stop codon of MT1-MMP by PCR to create an in-frame Bgl II site, which was ligated to the Bgl II site of GFP described above (pCEP4-MT1-GF
P). 293T cells are 0.2 mm thick glass bottom 35 mm diameter plates
(Matsunami Glass Ind., Ltd. Tokyo, Japan)
pCEP4-MT1-GFP (100ng), MMP-2 (100ng) and Claudi
An n-5 (100 ng) expression plasmid was introduced together. The total plasmid DNA was adjusted to 2 μg with pSG5 plasmid DNA. Cells are fixed with 4% paraformaldehyde and anti-MMP-2.
Monoclonal antibody (Fuji Chemical Industries Lt
d., Toyama, Japan) and sheep anti-Claudin-1 polyclonal antibody. TRITC-labeled anti-sheep IgG and Cy3-labeled anti-mouse IgG antibody were used as secondary antibodies. Fluorescence was monitored with an inverted confocal laser microscope (Carl Zeiss, Ind.). As a result of observation with a confocal laser microscope, MMP-2
Was not clearly immunostained in cells transfected with both MMP-2 and GFP cDNAs. Co-expression of MT1-GFP and MMP-2 caused a slight co-localization of MMP-2 and MT1-GFP on the cell surface. It was clearly enhanced by the introduction of TIMP-2. MMP-2 was weakly localized in the cytoplasmic granules of cells into which both MMP-2 and claudin-5 cDNA had been introduced. MT1-
The cell surface co-localization of GFP and MMP-2 was also stimulated by the expression of claudin-5, with MMP-2 and MT1-GFP at the cell border.
Co-localization was enhanced.

As described above, the present inventors
As a result of screening the expression of a gene that regulates MMP-2 activation mediated by 1-MMP, an endothelial cell-specific tight junc was identified as a factor that regulates MMP-2 activation mediated by MT1-MMP.
We succeeded in identifying claudin-5 which is an option (TJ) protein. And the factor is proMMP-2 by MT1-MMP
It was found that not only TMP-2 but also proMMP-2, which activates proMMP-2, promotes all previously reported MT-MMPs-mediated proMMP-2 activation.
Claudin-5 was identified as one of the factors that promote MT1-MMP-mediated proMMP-2 activation in 293T cells. Clau
In addition to din-5, the TIMP-2 gene was identified by the expression cloning method. Its expression showed a promoting effect similar to claudin-5 in 293T cells. In addition, some candidate genes can be cloned. The gene product had a positive or negative effect on MT1-MMP-mediated activation of proMMP-2.

Low level expression of endogenous TIMP-2 in 293T cells allows effective screening of factors involved in the regulation of MT1-MMP mediated proMMP-2 activation. The model of proMMP-2 activation by MT1-MMP is the formation of a ternary complex between proMMP-2, TIMP-2 and MT1-MMP, followed by TIMP flanked by proMMP-2 in this complex.
It is cleaved by -2-free MT1-MMP to form an active intermediate. Intermolecular auto-c of MMP-2 intermediate
Leavage gives rise to fully active MMP-2. Therefore, focal enrichment of MT1-MMP is a regulator for both steps of MMP-2 activation. Endogenous in 293T cells
The TIMP-2 concentration was suboptimal for proMMP-2 activation by MT1-MMP. Therefore, exogenous TI in 293T cells
Expression of MP-2 promotes proMMP-2 activation by MT1-MMP. Claudin-5 not only replaces TIMP-2 to promote MT1-MMP activation of proMMP-2 but also other MT-MMP
s-induced activation or cell-bound pr
It also induced activation by MT1-MMP mutants with poor or lacking oMMP-2 activating function. TIMP-2
Although an expression-enhancing effect was observed in MT1-MMP-mediated proMMP-2 activation, claudin-5-induced proMMP-2 activation by other MT-MMPs or MT1-MMP mutants was associated with TIMP-2 expression. Was inhibited by. These results show that TIMP-2 is MT-MMPs
Suggest that it is not involved in claudin-5-stimulated proMMP-2 activation. However, it does not completely eliminate the role of low concentrations of TIMP-2 in 293T cells.

TIMP-2-dependent activation of proMMP-2 by MT-MMPs was performed using a C-terminal domain-deficient proMMP-2 mutant (proΔMMP-2), which is believed to allow proMMP-2 to interact with TIMP-2. , More clearly shown. MT1-MM in 293T cells
Processing by P to form the active intermediate of proΔMMP-2 is most efficient without exogenous TIMP-2 expression and is dose-dependently inhibited by exogenous TIMP-2 expression.
However, MT1-MMP-induced proMMP-2 activation is conversely promoted by the expression of exogenous TIMP-2. These results are
TIMP-2-free MT1-MMP cleaves proΔMMP-2 to form an intermediate, and MT1-MMP-TIMP-2 complex that acts as a receptor / activator for proMMP-2 is proΔMMP-2
Suggest that it cannot be processed. This is not surprising. This is because proMMP-2 lacking the C-terminal domain can be activated by soluble MT1-MMP in the absence of TIMP-2 in solution. ΔMMP-2 intermediate is claudi
In the presence of n-5, it is processed to a more complete active form. Compared to 293T cells, COS-1 cells contain a high enough concentration of endogenous TIMP-2 for ternary complex formation and are mediated by MT1-MMP.
Promotes MMP-2 processing. Thus, in COS-1 cells, proMMP-2 activation by MT1-MMP was efficient, with low levels of exogenous TIMP-2 expression having no effect and high levels of inhibition. ProΔMMP-2 by MT1-MMP
Activation was barely seen in COS-1 cells. Expression of other MT-MMPs of native MT1-MMP and MT3-MMP and expression of MT1-MMP mutants was not shown in COS-1 cells.
The presence of claudin-5 also did not induce overt proMMP-2 processing. All this may be due to the high concentration of endogenous TIMP-2 in COS-1 cells.

Despite the binding of TIMP-2, proMMP-2 was processed to form the fully active form by MT-M.
Localized enrichment of MPs is required. For all MT-MMPs
catalytic domains are pr in a solution without TIMP-2
Since it is possible to cleave the Asn ⁶⁶ -Leu ⁶⁷ peptide bond of oMMP-2, they can be cleaved on the cell surface under appropriate conditions.
It should activate proMMP-2. Before that, MMP-2
Cell surface binding (which is mediated by ECM components, heparin and / or integrin αvβ3)
It plays a role in activation of proMMP-2. Clau
Din aggregates MT-MMPs and proMMP-2 and may induce not only the cleavage of the Asn ⁶⁶ -Leu ⁶⁷ bond but also the subsequent intermediate-to-active auto-cleavage.
Immunoprecipitation analysis showed that MT1-MMP and proMMP-2 were claudin-
Directly shown to interact with 1. In addition, a defect analysis revealed that the extracellular domain of claudin-1 is a catalyst of MT-MMPs.
Suggested to interact with cdomain. In addition, claud
The lack of C-terminal PDZ motif from in-1 did not affect the promoting effect, suggesting that claudin-1 binding to actin filaments through ZO-1, -2, and -3 is not essential. Another function of TIMP-2 for proMMP-2 activation is the stabilization of 57 kDa activated MT1-MMP, resulting in proMMP.
It may be MP-2 activation. However, claudin did not show such a stabilizing function.

All four claudin families tested promoted proMMP-2 activation in 293T cells. Claudi
n-5 / TMVCF is an endothelial cell-specific component of the TJ chain. And MT1-MMP plays an essential role in angiogenesis.
It is therefore speculated that claudin-5 regulates MT1-MMP activity during angiogenesis and / or the interaction of MT1-MMP with claudin affects TJ functions such as vascular permeability. Next, Claudin-1 is the most widely distributed TJ component, but the TJ localization of claudin-1 in MDCK epithelial cells is
Altered by transformation of cells with oncogne ras or by treatment with phorbol ester. COS-1 cells synthesize high levels of claudin-1. However,
It was not immunolocalized in the TJ chain, but was scattered and stained in the cell membrane and cytoplasm. The expression level of claudin-1 in 293T cells is much lower than that in COS-1 cells.

293T into which claudin-1 and MT1-MMP cDNA were introduced
In cells, claudin-1 and MT1-MMP were not only restricted to the TJ chain, but some of them were colocalized at sites other than the cell boundary. Co-localization of MMP-2 and claudin-1 was observed at the cell border and cytoplasm. Claudin-1
Was immunoprecipitated with MT1-MMP suggesting that part of the complex was not incorporated into the TJ chain. This is because claudin in the TJ chain is not easily solubilized in immunoprecipitation. Claudin- in the epididymis
Another study of 1 localization showed that claudin-1 expression was not exclusively localized to the TJ chain, but appeared along the entire interface of adjacent epithelial cells as well as along the basal plasma membrane. , Which suggests the role of claudin-1 as an adhesion molecule. These suggest that the claudin family proteins have a more motile function than that of the TJ chain. Tumor cell tip or invadopo
MT1-MMP targeting to dia is mediated by the transmembrane / cytoplasmic domain of MT1-MMP. But,
Claudin-induced proMMP-2 activation is a catalytic do of MT1-MMP
You only need main. This suggests that MT-MMP cell surface targeting and cell invasion to proMMP-2 activation is regulated by different mechanisms. Thus claudin is proMMP-2 by MT1-MMP
It was shown that upon activation, it not only activates proMMP-2 like TIMP-2, but it also promotes proMMP-2 activation by all MT-MMPs. Because of this, Clau
The interaction between din and MT-MMPs appears to regulate claudin function as well as pericellular MT-MMPs activity.

[0136]

The claudin family is involved in the activation of proMMP-2 by MT-MMPs (particularly MT1-MMP), so that a method for controlling and regulating proMMP-2 activation can be developed. ProMMP-2 via MT-MMPs by the claudins
Substances that inhibit activation, especially proMMP via MT1-MMP
-2 A substance that inhibits activation can be provided and developed.
-2. A method for controlling various physiological and biological processes resulting from activation of proMMP-2 using an activation inhibitor and a drug therefor can be provided. MT-MMPs, especially MT1-
It will contribute to the research and development of medicines and therapeutic methods related to the inhibition of cancer invasion and metastasis based on the MMP-mediated proMMP-2 activation regulation mechanism and angiogenesis. Obviously, the present invention may be carried out other than as specifically described in the above description and examples.
Many modifications and variations of the present invention are possible in light of the above teachings, and are therefore within the scope of the claims appended hereto.

[0137]

[Sequence list]                                SEQUENCE LISTING                                                                          <110> Fuji Yakuhin Kogyo Kabushiki Kaisha                                                                          <120> Claudin-Promoted Activation of Pro-MMP-2 Mediated by       MT-MMPs                                                                          <130> P-01NF358                                                                          <140> <141>                                                                          <160> 20                                                                          <170> PatentIn Ver. 2.0                                                                          <210> 1 <211> 211 <212> PRT <213> Homo sapiens                                                                          <400> 1 Met Ala Asn Ala Gly Leu Gln Leu Leu Gly Phe Ile Leu Ala Phe Leu   1 5 10 15                                                                          Gly Trp Ile Gly Ala Ile Val Ser Thr Ala Leu Pro Gln Trp Arg Ile              20 25 30                                                                          Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Ala Gln Ala Met Tyr Glu          35 40 45                                                                          Gly Leu Trp Met Ser Cys Val Ser Gln Ser Thr Gly Gln Ile Gln Cys      50 55 60                                                                          Lys Val Phe Asp Ser Leu Leu Asn Leu Ser Ser Thr Leu Gln Ala Thr  65 70 75 80                                                                          Arg Ala Leu Met Val Val Gly Ile Leu Leu Gly Val Ile Ala Ile Phe                  85 90 95                                                                          Val Ala Thr Val Gly Met Lys Cys Met Lys Cys Leu Glu Asp Asp Glu             100 105 110                                                                          Val Gln Lys Met Arg Met Ala Val Ile Gly Gly Ala Ile Phe Leu Leu         115 120 125                                                                          Ala Gly Leu Ala Ile Leu Val Ala Thr Ala Trp Tyr Gly Asn Arg Ile     130 135 140                                                                          Val Gln Glu Phe Tyr Asp Pro Met Thr Pro Val Asn Ala Arg Tyr Glu 145 150 155 160                                                                          Phe Gly Gln Ala Leu Phe Thr Gly Trp Ala Ala Ala Ser Leu Cys Leu                 165 170 175                                                                          Leu Gly Gly Ala Leu Leu Cys Cys Ser Cys Pro Arg Lys Thr Thr Ser             180 185 190                                                                          Tyr Pro Thr Pro Arg Pro Tyr Pro Lys Pro Ala Pro Ser Ser Gly Lys         195 200 205                                                                          Asp Tyr Val     210                                                                                                                                                   <210> 2 <211> 21 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 2 ccgaattctg cacctgccac c 21                                                                          <210> 3 <211> 23 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 3 tgaattcgac agatcacacg tag 23                                                                          <210> 4 <211> 33 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 4 aagcttgaat tcggcgcgcc agatatcgca tgc 33                                                                          <210> 5 <211> 32 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 5 cgccggcgcc atggcgatcg gctagcagat ct 32                                                                          <210> 6 <211> 24 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 6 caaggagtcg ccggcatagg agta 24                                                                          <210> 7 <211> 24 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 7 gccggcgact ccttgctgaa tctg 24                                                                          <210> 8 <211> 23 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 8 gactttgcag gacatccaca gcc 23                                                                          <210> 9 <211> 23 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 9 atgtcctgca aagtctttga ctc 23                                                                          <210> 10 <211> 25 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 10 cctggcattg ccataccatg ctgtg 25                                                                          <210> 11 <211> 25 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 11 tatggcaatg ccaggtacga atttg 25                                                                          <210> 12 <211> 21 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 12 ctctgcgaca cggaggacat c 21                                                                          <210> 13 <211> 21 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 13 ctgtggatgt cctccgtgtc g 21                                                                          <210> 14 <211> 21 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 14 tcaaagactt tggactggat c 21                                                                          <210> 15 <211> 21 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 15 ggcagatcca gtccaaagtc t 21                                                                          <210> 16 <211> 21 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 16 ccgaattctg cacctgccac c 21                                                                          <210> 17 <211> 23 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 17 gacggattcc acttagtctt tcc 23                                                                          <210> 18 <211> 23 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 18 caagcttggc atgaagtgta tga 23                                                                          <210> 19 <211> 23 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 19 tgaattcgac agatcacacg tag 23                                                                          <210> 20 <211> 31 <212> DNA <213> Artificial Sequence                                                                          <220> <223> Description of Artificial Sequence: Oligonucleotide       to act as a primer for PCR                                                                          <400> 20 ggagatctga cgtagttctt cttgtcgtag t 31

[Brief description of drawings]

FIG. 1 Human placental cDNA in human fetal kidney-derived 293T cells.
3 is an electrophoretic photograph of the results of gelatin zymography of the cell lysate obtained by transfection of the plasmid DNA from the library with MMP-9, MMP-2 and MT1-MMP. Processing of proMMP-2 to the active form is facilitated in lane 8.

FIG. 2 A. Co-transfection of control plasmid (pSG5) or claudin-5 plasmid, MMP-2 plasmid and each MT-MMPs plasmid into 293T cells, and gelatin zymography was performed on the obtained cell lysate. It is an electrophoresis photograph of the result. Claudin-5 is MT-
It is shown to promote proMMP-2 processing by MMPs. B. This is an electrophoretic photograph of the results obtained by subjecting the cell lysate used in the gelatin zymography of A above to SDS-PAGE under a reducing condition and examining the expression of MT-MMPs by Western blotting using each monoclonal antibody. In this figure, MT
The results of comparison with cells into which both 1-MMP and TIMP-2 genes have been introduced are also shown. It is shown that the expression of MT-MMPs is not affected by the expression of claudin-5.

Figure 3 A. Co-transfection of control plasmid (pSG5) or claudin-5 plasmid with MMP-2 plasmid and wild type MT1-MMP or its mutant plasmid as shown in 293T cells. 3 is an electrophoretic photograph of the result of analyzing the cell lysate obtained by gelatin zymography. Claudin-5 due to MT1-MMP mutant
It has been shown to promote proMMP-2 processing. B. The cell lysate used for gelatin zymography in A. above was subjected to SDS-PAGE under reducing conditions and MT1-MMP
3 is an electrophoretic photograph showing the results of examining the expression of mutant Δ535 or Δ335 by Western blotting using a monoclonal antibody against MT1-MMP.

FIG. 4 shows proΔMMP-2 plasmid as control plasmid (pSG5), MT1-MMP plasmid and claudin-5
FIG. 3 is an electrophoretic photograph showing the result of gelatin zymography analysis of the resulting cell lysate obtained by cotransfection of 293T cells with a plasmid. It is shown that Claudin-5 promotes processing of proΔMMP-2 by MT1-MMP.

FIG. 5: A and B. ProMMP-2 or proΔMMP-2 plasmid and MT1-MMP were co-transfected with 293T cells (A) or COS-1 cells (B) together with TIMP-2 plasmid in the amounts shown in the figure. It is the electrophoresis photograph of the result of having analyzed the cell lysate obtained by transfecting the gene by gelatin zymography. C. Control plasmid or claudin-5 plasmid, proMMP-2 and MT1-MMP mutant Δ535 were co-transfected into 293T cells together with TIMP-2 plasmid in the amount shown in the figure, and the resulting cell lysate was used. It is an electrophoretic photograph of the result analyzed by gelatin zymography.

FIG. 6: MMP-2 and MT1-MMP plasmids with control or claudin plasmids as shown
It is an electrophoretic photograph of the result of analyzing the obtained cell lysate by cogene transfer to 293T cells by gelatin zymography (upper panel). The obtained cell lysate was also subjected to SDS-PAGE under reducing conditions, and claudin was obtained by Western blotting using the antibody as shown in the figure.
3 is an electrophoretic photograph of the results of examining the presence of (lower panel).

[Figure 7] A: MT1-MMP (upper panel) or Δ335
(Lower panel) Control plasmid (lane C), claudin-1 (lane
W) or the mutant plasmids shown in the figure, and electrophoretically taken as a result of analyzing the cell lysate obtained by transducing 293T cultured cells for 48 hours and performing gelatin zymography. The extracellular domain of claudin-1 is MT1-MMP
It is shown to be essential for the promotion of proMMP-2 activation via the. B: The cell lysate used for gelatin zymography in A above was subjected to SDS-PAGE under reducing conditions, and claudin- was analyzed by Western blotting using an antibody against claudin-1.
1 is an electrophoretic photograph showing the results of examining the presence of a mutant.

FIG. 8: Schematic diagram of C: claudin-1 mutant The putative transmembrane domain was darkened. E1, first extracellular domain, E2, second extracellular domain, C, cysteine, S, serine.

FIG. 9 is an electrophoretic photograph of co-immunoprecipitates of Claudin-1, MT1-MMP and MMP-2. 293T cells were transduced with claudin-1 plasmid alone, MT1-MMP plasmid alone, MMP-2 plasmid alone or a plasmid as shown in the figure. Twenty-four hours after transduction, cells were labeled with S-35 methionine and cysteine for 4 hours. Cells were solubilized in cell lysate buffer, immunoprecipitated with anti-MT1-MMP monoclonal antibody, anti-MMP-2 monoclonal antibody or anti-claudin-1 polyclonal antibody and adsorbed with Protein G-Sephadex beads, Recovered. Immunoprecipitates were analyzed by 12% SDS-PAGE analysis under reducing conditions and detected by bioimage analyzer.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 11/00 A61P 29/00 101 4C084 19/02 35/00 4H045 29/00 101 C07K 14/705 35/00 16 / 28 C07K 14/705 C12N 1/15 16/28 1/19 C12N 1/15 1/21 1/19 9/64 1/21 9/99 5/10 C12Q 1/02 9/64 G01N 33/15 Z 9 / 99 33/50 Z C12Q 1/02 C12N 15/00 ZNAA G01N 33/15 5/00 A 33/50 A61K 37/02 (72) Inventor Kenichi Obata 530 Chokeiji Temple, Takaoka City, Toyama Prefecture Fuji Yakuhin Kogyo Co., Ltd. the internal F-term (reference) 2G045 AA34 AA35 BB20 CB01 DA36 FB02 FB03 4B024 AA01 BA41 CA01 GA11 4B050 CC10 DD07 HH01 KK18 4B063 QA05 QA20 QQ21 QQ41 QQ61 QR16 QR77 QS24 QS31 QX01 4B065 AA93Y AB01 AC14 BA02 CA24 CA44 CA60 4C084 AA02 AA07 AA13 AA17 DC50 NA14 ZA452 ZA592 ZA962 ZB152 ZB262 4H045 AA10 AA11 DA55 EA20 FA74

Claims (40)

[Claims] 1. A method for activating latent matrix metalloprotease-2 (proMMP-2) via membrane-type matrix metalloproteases (MT-MMPs) by a member selected from the claudin family. 2. Claudin-1 (claudin-1) and claudin-2 are selected from the claudin family.
(claudin-2), claudin-3 (claudin-3), or claudin-5 (claudin-5). 3. Membrane-type matrix metalloprotease-1
(membrane-type 1matrix metalloproteinase: MT1-MM
The method according to claim 1 or 2, which is activation of proMMP-2 via P). 4. ProMMP-2, which is characterized by inhibiting the complex formation between (1) one selected from the claudin family and (2) one selected from MT-MMPs and proMMP-2.
Activation inhibitor. 5. The method according to claim 4, which inhibits complex formation between (1) one selected from the claudin family and (2) one selected from MT1-MMP and proMMP-2. proMMP-2 activation inhibitor. 6. (i) inhibiting the formation of a complex between one selected from the claudin family and one selected from MT-MMPs to inhibit the activation of proMMP-2, or (i
i) selected from claudin family and proMMP
An inhibitor of angiogenesis, cell migration, invasion and / or metastasis, which inhibits the formation of a complex with -2 to inhibit the activation of proMMP-2. 7. The agent according to claim 6, wherein the cell is a cancer cell. 8. The complex is a complex of (1) one selected from the claudin family and (2) one selected from MT1-MMP and proMMP-2.
Or the agent of 7. 9. The complex formation is performed at least with an extracellular domain of one selected from the claudin family.
8. The agent according to any one of 8. 10. ProMMP-2, which inhibits complex formation between (1) one selected from the claudin family and (2) one selected from MT-MMPs and proMMP-2.
A method of inhibiting activation. 11. The method according to claim 1, which inhibits complex formation between (1) one selected from the claudin family and (2) one selected from MT1-MMP and proMMP-2.
The method described in 0. 12. (i) inhibits the complex formation between one selected from the claudin family and one selected from MT-MMPs to inhibit the activation of proMMP-2, or
(ii) selected from claudin family and proM
A method for inhibiting angiogenesis, cell migration, invasion and / or metastasis, which comprises inhibiting complex formation with MP-2 to inhibit activation of proMMP-2. 13. The method according to claim 12, wherein the cell is a cancer cell. 14. The complex is a complex of (1) one selected from the claudin family and (2) one selected from MT1-MMP and proMMP-2. Or 13 methods. 15. The complex formation is performed at least with an extracellular domain selected from the claudin family.
The method according to any one of 0 to 14. 16. A substance having an activity of inhibiting the complex formation between (1) one selected from the claudin family and (2) one selected from MT-MMPs and proMMP-2. 17. The complex is a complex with (a) a cell surface selected claudin family or (b) a cell surface selected MT-MMPs. The substance according to claim 16, characterized in that 18. (A) A claudin selected from the claudin family and having a mutation in its extracellular domain region, a derivative thereof, an analogue thereof, or an activity substantially equivalent thereto. (B) a peptide fragment selected from the claudin family and corresponding to a part of a peptide corresponding to the extracellular domain region thereof, a derivative having substantially the same activity as those, or an analog thereof; The substance according to claim 16 or 17, which is selected from the group consisting of (C) an antibody against the extracellular domain region of the claudin family. 19. (A) The mutation in the extracellular domain region of the one selected from the claudin family is selected from the group consisting of deletion, substitution and insertion of amino acid residues; (B) (i The peptide corresponding to the extracellular domain region has the amino acid sequence Asp ^{38 to} Phe ⁶⁷ of human claudin-1 shown in SEQ ID NO: 1 in the sequence listing.
Amino acid sequence of human claudin-1 corresponding to the peptide Val ^{55 to} Cys ⁶⁴
Peptide corresponding to the amino acid sequence of human claudin-1 Asn ¹⁴² ~ Val
Selected from the group consisting of peptides corresponding to ¹⁵⁵ and (ii)
The peptide having at least 3 or more consecutive amino acid residues among the peptides; or (C) (i) the peptide corresponding to the extracellular domain region is the human claudin described in SEQ ID NO: 1 of the Sequence Listing. -1 amino acid sequence Asp ^{38 to} Phe ⁶⁷
Amino acid sequence of human claudin-1 corresponding to the peptide Val ^{55 to} Cys ⁶⁴
Peptide corresponding to the amino acid sequence of human claudin-1 Asn ¹⁴² ~ Val
Selected from the group consisting of peptides corresponding to ¹⁵⁵ and (ii)
The substance according to any one of claims 16 to 18, which is an antibody against one having at least 3 or more consecutive amino acid residues among the peptides. 20. (i) A nucleic acid encoding the one described in (A) of claim 18, (ii) a peptide corresponding to a part of the peptide corresponding to the extracellular domain region of (B) of claim 18. A nucleic acid encoding a fragment, (iii) a nucleic acid capable of hybridizing with the nucleic acid of (i) above, and (iv) the above (i) to
A nucleic acid selected from the group consisting of nucleic acids having substantially the same activity as any one of the nucleic acids of (iii). 21. (i) A nucleic acid encoding the mutant claudin according to claim 19 (A), a derivative thereof or an analogue thereof, or a nucleic acid having an activity substantially equivalent to them,
i) a nucleic acid encoding the peptide fragment according to (B) of claim 19, (iii) a nucleic acid capable of hybridizing with the nucleic acid according to (i) above, and (iv) any of (i) to (iii) above 21. The nucleic acid according to claim 20, which is selected from the group consisting of nucleic acids having substantially the same activity as one nucleic acid. 22. A mutant lacking amino acid residues at positions 38-67 of human claudin-1 (M1Δ38-67), and a mutant lacking amino acid residues at positions 55-64 of human claudin-1. Body (M2
Δ55-64), a mutant lacking amino acid residues 142-155 of human claudin-1 (M3Δ142-155), the cysteine residue at position 54 of human claudin-1 was replaced with a serine residue Mutant (M4C54S), mutant in which the cysteine residue at position 64 of human claudin-1 was replaced with a serine residue (M5C64S), mutant in which the PDZ motif on the C-terminal side of human claudin-1 was destroyed
(M6ΔYV) and 1-101 on the N-terminal side of human claudin-1
22. An enzyme selected from the group consisting of mutants (M7Δ1-101) in which the amino acid residue at position 1 is deleted, or those having an activity substantially equivalent to those selected from the group, 20 or 21. Nucleic acid. 23. A vector containing the nucleic acid according to any one of claims 20 to 22. 24. A transformant containing the nucleic acid according to any one of claims 20 to 22 or the vector according to claim 23. 25. A gene therapy agent comprising the nucleic acid according to any one of claims 20 to 22 or the vector according to claim 23. 26. A medicinal or veterinary medicine containing the compound according to any one of claims 4 to 9 or the compound according to any one of claims 16 to 24. 27. The medicine or veterinary medicine according to claim 26, which is for treating and / or preventing a disease or a pathological condition caused by proMMP-2 activation. 28. The medicine or veterinary medicine according to claim 26, which is for suppressing and / or preventing angiogenesis, cell migration, invasion and / or metastasis. 29. The medicine or veterinary medicine according to claim 28, wherein the cell is a cancer cell. 30. Inhibition of proMMP-2 activation using (1) a complex selected from the claudin family and (2) one selected from MT-MMPs and proMMP-2 as an index. A screening method comprising screening a substance. 31. The method according to claim 3, which inhibits complex formation between (1) one selected from the claudin family and (2) one selected from MT1-MMP and proMMP-2.
The method described in 0. 32. (1) those selected from the claudin family, (2) those selected from MT-MMPs, and (3) proM.
32. The method according to claim 30 or 31, characterized in that a cell line expressing MP-2 is used. 33. A substance which inhibits the activation of proMMP-2 obtained by the method according to any one of claims 30 to 32. 34. (i) inhibiting the formation of a complex between one selected from the claudin family and one selected from MT-MMPs to inhibit proMMP-2 activation, or
(ii) selected from claudin family and proM
Angiogenesis, cell migration, invasion and // inhibition of proMMP-2 activation by inhibiting complex formation with MP-2
Alternatively, a screening method comprising screening a substance having an activity of inhibiting metastasis. 35. (1) those selected from the claudin family, (2) those selected from MT-MMPs, and (3) proM.
35. The method of claim 34, wherein a cell line expressing MP-2 is used. 36. A substance having angiogenesis, cell migration, invasion and / or metastasis inhibitory activity obtained by the method according to claim 34 or 35. 37. In a screen for inhibition of proMMP-2 activation, (i) one selected from the claudin family, (2) one selected from MT-MMPs, and (3) proM.
In the presence of MP-2, (ii) the degree of activation of proMMP-2 in the coexistence of a candidate compound for inhibiting proMMP-2 activation or (iii) in the absence of the candidate compound for inhibiting the activation of proMMP-2 And a method for screening a substance that inhibits the activation of proMMP-2. 38. (1) those selected from the claudin family, (2) those selected from MT-MMPs, and (3) proM.
38. The method of claim 37, wherein a cell line expressing MP-2 is used. 39. The method according to claim 37 or 3, wherein (1) one selected from the claudin family and (2) MT1-MMP and (3) proMMP-2 are screened.
8. The method according to 8. 40. A substance which inhibits the activation of proMMP-2 obtained by the method according to any one of claims 37 to 39.