JP2005154389A - Antibody discriminating shape of cd44 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody that can elucidate the complicated phenomena, and provide tools/technics for studying/elucidating the functions of CD44, because CD44 is a cell adhesion molecule that works as a main receptor to hyaluronic acid and concerns to a variety of biological and pathological processes, for example, homing of lymphocyte cells, activation of T-cells, wound healing, neovascularization or the metastatic extension of tumor cells, but the detail analyses has been impossible because extreme complication of the molecules and this invention solves these problems. <P>SOLUTION: Some antibody recognizing the characteristic sequence in the CD44 stem area is influenced in its bonding reaction by the saccharide chain modification occurring on the CD44 molecule, particularly by the O-bonding type saccharide chain modification or the modification near the recognition site. Thus, these antibodies are useful for detecting and analyzing the specific saccharide modification at the CD 44 site, particularly detecting and analyzing still unknown rolls in tumors. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to antibodies that make it possible to distinguish the form of CD44. The present invention relates to a technique for discriminating a sugar chain on a CD44 molecule by discriminating the characteristic CD44 form with the specific antibody, or discriminating between tumor cells and non-tumor cells in a tumor tissue or the like. .

  Hyaluronic acid (HA) is a glycosaminoglycan that retains a large amount of water in the extracellular matrix that fills many tissue gaps and is used in many organisms such as inflammation, wound healing, tissue remodeling and cell motility (Non-Patent Document 1: Lee et al., Curr Opin Cell Biol, 12, 581-586, 2000). HA is detected at a high concentration in a tumor having a high proliferation ability and invasive ability, and is considered to be useful for confirming the state of the tumor (Non-patent Document 2: Toole, et al., J Biol). Chem, 277, 4593-4596, 2002). It is known that many tumors synthesize HA and release it to the surrounding environment (Non-Patent Document 3: Knudson et al., Front Biosci, 3, D604-615, 1998). It has been shown that HA production by tumor cells has a great influence on their malignancy using non-HA synthesis deficient cells (Non-patent Document 4: Itano et al., Proc Natl Acad Sci USA, 99, 3609). -3614, 2002). This effect is converted into multiple signals via cell surface receptors such as CD44, LYVE-1, Layilin and RHAMM (Non-Patent Document 5: Entwistle et al., J Cell Biochem, 61: 569-577, 1996).

CD44 is a type I membrane protein that has been identified as a surface antigen of lymphocytes, and has subsequently been reported to be expressed in many cell lines such as fibroblasts, epithelial cells, and cancer cells (non-patented). Reference 6: Naot et al., Adv Cancer Res, 71, 241-319, 1997). CD44 is a major receptor for hyaluronan and plays an important role in physiological and pathological processes such as lymphocyte homing, T cell activation, wound healing, angiogenesis and metastatic spread of cancer cells. (Non-Patent Document 6: Naot et al., Adv Cancer Res, 71, 241-319, 1997). CD44 can also bind to fibronectin, collagen and the like.
CD44 is a single transmembrane glycoprotein consisting of four functional domains: (1) outer extracellular domain, (2) inner extracellular domain, (3) transmembrane domain (TM) And (4) has an intracellular domain. The gene for CD44 is said to be encoded by 20 exons (previously 19 were encoded by exons, of which exon 6 was reclassified as exon 6a and exon 6b, now exon 6 and exon 7 ), Exons 6-15 are called variable exons (correspondingly referred to as v1-10), and have undergone extensive reconfiguration by variable spons and alternative splicing. It is thought to produce numerous isoforms. Alternative splicing also occurs in the exons that encode the intracellular domain of CD44, namely exon 19 and exon 20, resulting in short intracellular CD44 and full-length CD44 of the intracellular domain. ing.

  The standard and most widely distributed CD44 protein is CD44H, which consists of proteins encoded by exons 1-5, 16-18 and 20, and the extracellular domain of CD44H is exons 1-5, 16 And 17 are encoded predominantly, the transmembrane domain is encoded primarily by exon 18, and the intracellular domain is encoded by exon 20. CD44H is composed of 341 amino acids (aa) excluding 20 amino acid residues of the signal peptide of the amino acid sequence represented by SEQ ID NO: 1, of which 248 aa is the extracellular position and 72 aa is the intracellular site. It is composed. The N-terminal 109 aa has 6 cysteine residues and forms a globular region for ligand binding (Non-Patent Document 6: Naot et al., Adv Cancer Res, 71, 241-319, 1997). ). The 139 aa stem (stem or axis) region located between the Globular region and the cell membrane contains a selective splicing insertion site, a glycosylation site, and a cleavage site for Shedding. . Following the 21 aa type I transmembrane region, it has a 72 aa intracellular region that functions as a binding site for various proteins such as ERM (ezrin, radixin and moesin) proteins and ankyrins that bind to the actin skeleton ( Non-Patent Document 6: Naot et al., Adv Cancer Res, 71, 241-319, 1997). The regulatory functions of low molecular weight GTP-binding proteins such as Rho-GDI and Tiam1 are also included in this site (Non-patent Document 7: Bourguignon et al., J Biol Chem, 275, 1829-1838, 2000). Thus, the extracellular part of CD44 mediates cell-cell and cell-matrix interactions while having a tissue structure with relatively soft adhesion, and the intracellular part interacts with the actin skeleton. Involved in intracellular signal transduction and control of cell morphology.

  CD44 has many isomers generated by alternative splicing (non-patent document 6: Naot et al., Adv Cancer Res, 71, 241-319, 1997). The most quantitative and basic form is the standard CD44 (CD44H or CD44s). Alternative splicing inserts additional sequences encoded by a special exon into the stem region. So far, transcripts with at least 20 alternatively spliced exons have been detected, and each isomer is thought to have specific functions, such as differences in HA recognition ability. (Non-patent document 8: Stamenkovic et al., Embo J, 10: 343-348, 1991). Furthermore, many relationships between these isomers and cancer have been reported. For example, expression of isomers having variant sequences v6 and v8-v10 is correlated with metastatic spread of cancer cells (Non-patent Document 9: Gunthert et al., Cell, 65, 13-24, 1991). MMP-7, one of matrix metalloproteases, has been reported to bind to heparan sulfate proteoglycan (HSPG) modified to variant sequence v3 (Non-Patent Document 10: Yu et al., Genes Dev., 16, 307-323, 2002).

CD44 undergoes extensive post-translational modifications such as phosphorylation, glycosylation, and addition of glycosaminoglycan chains found in several isomers, as found in transmembrane glycoprotein receptors. Posttranslational modifications, such as particularly variable N-linked and O-linked sugar chains, give rise to further diversity of isomers (Non-Patent Document 11: Goldstein et al., Cell, 56, 1063-1072, 1989). ). CD44H consists of a polypeptide estimated to have a molecular weight of 37 kDa, but the observed molecular weight is about 85-95 kDa due to many sugar chain modifications. There are six N-linked glycosylation sites and a number of O-linked glycosylation sites on the CD44H molecule. Most N-linked glycosylation sites are present in the N-terminal Globulular region involved in binding to HA, and it is thought that the HA binding ability is controlled by the modification mode. In addition, the four serine-glycine motifs in the Stem region are modifiable sites for proteoglycan heparan sulfate and chondroitin sulfate. Addition of these sugar chains is necessary for HA binding, but it is known that, depending on the modification mode, HA binding is conversely inhibited. For example, it has been reported that inhibition of N-linked sugar chain modification by tunicamycin reduced HA binding ability (Non-patent Document 12: Cattera et al., Clin. Exp. Metastasis, 17, 583-591, 1999) There is also a report that non-patent document 13: Jones et al., Int. J. Biochem. Cell Biol., 35, 1361-1377, 2003) There is also a report that non-patent document 14: Katoh et al., J. Exp. Med., 182, 419-429, 1995) that HA binding ability was improved by removing sialic acid by neuraminidase treatment. Perhaps the balance of various glycosylation modifications regulates the strength of HA binding. In addition, this balance of HA binding is thought to change the motility and morphology of cells, and also affect the invasion and metastasis of cancer cells. However, it is not easy to detect a CD44 sugar chain at a specific site because of the variety of sugar chain modifications and the number of sites that can be added. Therefore, it is very difficult to clarify the biological role of site-specific sugar chains (particularly modifications in the stem region).
Many antibodies against CD44 have been produced so far. Its properties include not only recognizing the CD44 molecule, but also inhibiting its ability to bind to HA or conversely inducing it. The antibody described in Non-Patent Document 15: Martegani et al., Am. J. Pathol., 154, 291-300, 1999 is an antibody that recognizes the exon region of CD44 isomer or an antibody that recognizes the constant region. It has the property that reactivity is inhibited by the combination of exon sequence and post-translational modification. Modifications that affect this are N-linked sugar chains and glycosaminoglycan side chains. These sugar chain addition sites are easily predicted from the CD44 amino acid sequence, and many analyzes of the function of the sugar chain have been reported.

Lee et al., Curr Opin Cell Biol, 12, 581-586, 2000 Toole, et al., J Biol Chem, 277, 4593-4596, 2002 Knudson et al., Front Biosci, 3, D604-615, 1998 Itano et al., Proc Natl Acad Sci U S A, 99, 3609-3614, 2002 Entwistle et al., J Cell Biochem, 61: 569-577, 1996 Naot et al., Adv Cancer Res, 71, 241-319, 1997 Bourguignon et al., J Biol Chem, 275, 1829-1838, 2000 Stamenkovic et al., Embo J, 10: 343-348, 1991 Gunthert et al., Cell, 65, 13-24, 1991 Yu et al., Genes Dev., 16, 307-323, 2002 Goldstein et al., Cell, 56, 1063-1072, 1989 Cattera et al., Clin. Exp. Metastasis, 17, 583-591, 1999 Jones et al., Int. J. Biochem. Cell Biol., 35, 1361-1377, 2003 Katoh et al., J. Exp. Med., 182, 419-429, 1995 Martegani et al., Am. J. Pathol., 154, 291-300, 1999

  Among the sugar chain modifications in CD44, O-linked sugar chains have been reported to have effects on hyaluronan binding and their relationship with cancer cell growth, invasion and metastasis. Because of the large number of sites that can be added due to the absence of a specific sequence, it is very difficult to analyze the function, particularly the function of a sugar chain at a specific site. In addition, there have been many reports on the relationship between isomers and diseases, and many analyzes using antibodies have been conducted. The relationship between glycosylation and diseases, especially cancer, etc., is analyzed by antibody reactivity. There were no reports revealed by.

Although the CD44 stem region is present in common in the CD44 molecule, the present inventors are sites where alternative splicing (conjugation) occurs at that site, and also the sugar chain modification site and Shedding (cleavage / release). In view of the fact that it contains a cleavage site, an antibody that specifically recognizes a specific epitope of the stem region is thought to contribute to solving the above-mentioned problems, and as a result of intensive research, there are specific features of the CD44 stem region. We have succeeded in producing antibodies (including monoclonal antibodies) that are specifically reactive (or recognize) to sequences and characteristic structures. By using the specific antibody, the inventors have succeeded in finding that a characteristic structure appearing in the CD44 molecule can be analyzed, and completed the present invention.
Typically, we have monoclonal antibodies (285-2F12, 284-23F1, 268-1F5, 294-6F2) and polyclonal antibodies (C-6) that recognize epitopes with a limited range of the CD44 stem region. Succeeded in producing. Furthermore, we succeeded in identifying the recognition site of each antibody in more detail using the amino acid sequence deletion mutant of the CD44 stem region, and as a result, all antibodies were directed against polypeptides prepared in E. coli. We also succeeded in confirming the reactivity by Western blot. Furthermore, when the reactivity of each antibody to endogenous CD44 molecules expressed in several tumor cell lines was examined, 285-2F12 and 284-43F1 reacted with all CD44 molecules, but 268-1F5 and 294 -6F2 was not reactive to some CD44 molecules. Since these CD44H molecules were considered to have full-length peptides due to their molecular weight, it was speculated that the inhibition of antibody reactivity was due to post-translational modification. Thus, CD44H with post-translational modifications equivalent to the endogenous CD44H molecule was expressed in ZR-75-1 cells, and the molecule was analyzed using techniques such as deglycosylation and amino acid substitution mutants. As a result, the present inventors have confirmed that the O-linked sugar chain modification inhibits the reactivity by blocking the recognition site of the antibody.

In one embodiment, the present invention provides:
[1] An anti-CD44 antibody characterized by specifically recognizing a site consisting of a continuous amino acid sequence present in the inner extracellular domain of the extracellular domain of CD44 protein (provided by SEQ ID NO: 1 Except for an anti-CD44 antibody characterized by specifically recognizing a continuous amino acid sequence site of Thr ¹⁶³ -Arg ¹⁸⁶ present in the detected amino acid sequence).
[2] The above-mentioned [1], wherein the inner extracellular domain is a stem region of the extracellular domain and a region continuous with the region immediately before the transmembrane domain (TM). The described anti-CD44 antibody.
[3] The inner extracellular domain contains amino acid residues up to Cys ¹²⁹ on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1 of CD44H protein (including the 20 amino acid residues of the signal peptide). The continuous amino acid sequence found in the region from the amino acid residue of Thr ¹³⁰ adjacent to the globular region consisting of the group to the 268th amino acid residue Glu counting from the N-terminal side. The anti-CD44 antibody according to [1] or [2].
[4] The above-mentioned [1], wherein the portion consisting of a continuous amino acid sequence present in the inner extracellular domain among the extracellular domains of CD44 protein is at least 2 to 159 amino acid residues. Anti-CD44 antibody.
[5] The above-mentioned [1], wherein the portion consisting of a continuous amino acid sequence present in the inner extracellular domain among the extracellular domains of CD44 protein is at least 4 to 50 amino acid residues. Anti-CD44 antibody.
[6] Specific recognition of the continuous amino acid sequence found in the region immediately before the transmembrane domain (TM) in the stem region and the contiguous region of CD44 protein, and being sensitive to sugar chain modification An anti-CD44 antibody characterized by
[7] The anti-CD44 antibody according to [6] above, wherein the sugar chain modification of the CD44 protein is an O-linked sugar chain modification.
[8] The anti-CD44 antibody according to [6] or [7] above, wherein the sugar chain modification of the CD44 protein is an O-linked sugar chain modification with sialic acid.
[9] The anti-CD44 antibody according to any one of [6] to [8] above, wherein binding to the CD44 protein is inhibited by modification of the sugar chain of the CD44 protein.
[10] The anti-CD44 antibody according to any one of [6] to [9] above, which is reactive with tumor cells but non-reactive with stromal cells in a cancer tissue.
[11] Recognizing Pro ^220- Pro ²³⁰ of the amino acid sequence represented by SEQ ID NO: 1 of CD44H protein (counting including 20 amino acid residues of the signal peptide) [10] The anti-CD44 antibody according to any one of [10].
[12] The contiguous amino acid sequence of Ser ²³⁴ -Thr ²³⁷ of the amino acid sequence represented by SEQ ID NO: 1 of CD44H protein (including the 20 amino acid residues of the signal peptide) allows binding to CD44 protein. The anti-CD44 antibody according to [6] above, which is inhibited.
[13] The above [6], which recognizes Asp ²⁴³ -Gln ²⁵⁰ of the amino acid sequence represented by SEQ ID NO: 1 of CD44H protein (counted including 20 amino acid residues of the signal peptide) [12] The anti-CD44 antibody according to [12].
[14] The anti-CD44 antibody according to [13] above, wherein the reactivity with the CD44 protein is changed by sugar chain modification in the vicinity of the epitope.
[15] The anti-CD44 antibody according to any one of [1] to [5] above, which is not affected by O-linked sugar chain modification of CD44 protein.
[16] CD44 protein SEQ ID NO: above, characterized in that to recognize Pro ²⁰⁵ -Ile ²¹⁹ in 1 represented by the amino acid sequence (counting, including 20 amino acid residues of the signal peptide) (1) to The anti-CD44 antibody according to any one of [5] and [15].
[17] CD44 protein SEQ ID NO: above, characterized in that to recognize Thr ¹³⁰ -Arg ¹⁶² of 1 represented by the amino acid sequence (counting, including 20 amino acid residues of the signal peptide) (1) to [5] The anti-CD44 antibody according to any one of [5].
[18] The anti-CD44 antibody according to any one of [1] to [17] above, wherein the antibody is a monoclonal antibody.
[19] A method for discriminating a CD44 sugar chain, wherein a difference in the sugar chain of CD44 expressed on a cell is detected using the anti-CD44 antibody according to any one of [1] to [17].
[20] A method for discriminating a cell sugar chain in a tumor tissue, wherein a difference in sugar chain modification of the cell in the tumor tissue is detected using the anti-CD44 antibody according to any one of [1] to [17] .
[21] The method described in [20] above, wherein the cell is a tumor cell.
[22] A method of discriminating tumor cells and non-tumor cells in a tumor tissue using the anti-CD44 antibody according to any one of [1] to [17].
[23] The method described in [22] above, wherein the non-tumor cell is a stromal cell.
[24] containing the anti-CD44 antibody according to any one of [1] to [17] as an active ingredient,
(1) Detecting the difference in the sugar chain of CD44 expressed on cells,
(2) detecting differences in cell glycosylation in tumor tissue, and
(3) An anti-CD44 antibody reagent which is used for being selected from the group consisting of: distinguishing tumor cells and non-tumor cells in tumor tissue.
[25] The anti-CD44 antibody according to any one of the above [1] to [17], which is solid-phased.
[26] The anti-CD44 antibody according to any one of [1] to [17] above, which is labeled with a detectable label.
[27] At least the following steps:
(a) contacting the test biological sample with a carrier on which the anti-CD44 antibody according to any one of the above [1] to [17] is immobilized;
(b) washing the solid phase carrier brought into contact with the biological sample;
(c) a step of contacting an anti-CD44 antibody labeled with a solid phase carrier contacted with the biological sample and recognizing an epitope different from the immobilized anti-CD44 antibody;
(d) measuring a solid-phased label or a label that is free; and
(e) The labeled amount measured in step (d) is used as an index of CD44 amount,
(1) Detecting the difference in the sugar chain of CD44 expressed on cells,
(2) detecting differences in cell glycosylation in tumor tissue, and
(3) A step of distinguishing between tumor cells and non-tumor cells in a tumor tissue (however, in place of the anti-CD44 antibody described in any one of [1] to [17] above) (c) Using the carrier on which the anti-CD44 antibody is immobilized, the same treatment as in the above step (a) is carried out. On the other hand, in place of the anti-CD44 antibody of (c) above, (Including cases where the same treatment as in the above step (c) is performed using an antibody labeled with any one of the anti-CD44 antibodies)
A method for measuring a biological activity phenomenon associated with CD44, comprising:
[28] The method according to [27] above, wherein the antigen amount is quantitatively measured.
[29] At least the following steps:
(a) subjecting the test biological sample to tissue immobilization treatment;
(b) a step of using the tissue-fixed specimen prepared in step (a) as a section;
(c) a step of subjecting the sectioned tissue to immunohistological staining with the anti-CD44 antibody according to any one of [1] to [17] above;
(d) Using the degree of immunohistochemical staining in the test biological sample as an index,
(1) Detecting the difference in the sugar chain of CD44 expressed on cells,
(2) detecting differences in cell glycosylation in tumor tissue, and
(3) A method for measuring a biological activity phenomenon related to CD44, characterized in that it is selected from the group consisting of: distinguishing tumor cells and non-tumor cells in tumor tissue.
[30] An array for molecular analysis of CD44, wherein at least one or more of the anti-CD44 antibodies according to any one of [1] to [17] are arranged on a matrix array for protein analysis.
The amino acid sequence of the polypeptide encoded by the CD44H cDNA is shown in SEQ ID NO: 1 and FIG. 5.The 20 residues on the N-terminal side are considered to be signal peptides, and the CD44H protein is Gln (Q) It is considered to consist of 341 amino acid residues starting with.

A CD44 stem that contains a glycosylation site and a cleavage site for Shedding (cleavage / release), even though it is common to CD44 molecules, where alternative splicing occurs. Antibodies that specifically recognize specific epitopes in the region are available. Due to the successful production of antibodies (including monoclonal antibodies) specifically reactive (or recognizing) specific characteristic sequences and characteristic structures of the CD44 stem region, The characteristic structure that appears can be analyzed. It is possible to monitor the glycosylation state of specific sites occurring in the CD44 molecule, and it can be used to study the function and regulation mechanism of these sugar chains. Help to reveal the enzyme. By using the antibody having such very characteristic properties, the O-linked sugar chain-modifying properties of the CD44 molecule can be clarified, and further, tumor cells can be identified by the difference in reactivity. This antibody makes it possible to analyze the CD44 fragment produced by the protease. The antibodies of the present invention are useful for detecting CD44 fragments cleaved by certain enzymes, such as MT1-MMP, and can be used to reveal their interactions. This antibody is useful as a powerful research tool for the sugar chain function and regulatory mechanism of the CD44 molecule.
Other objects, features, excellence 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 following description and the description of specific examples and the like, show preferred embodiments of the present invention and are presented only for explanation. I want. Various changes and / or modifications (or modifications) within the spirit and scope of the present invention disclosed herein will occur to those skilled in the art based on the following description and knowledge from other parts of the present specification. Will be readily apparent. All patent documents and references cited herein are cited for illustrative purposes and are not to be construed as a part of this specification. is there.

While the present invention is common to CD44 molecules, it is a site where alternative splicing (conjugation) occurs at the site, and includes a glycosylation site and a cleavage site for Shedding (cleavage / release). Antibodies that specifically recognize specific epitopes in the stem region, in particular antibodies that specifically react (or recognize) specific sequences and structures in the CD44 stem region (including monoclonal antibodies) ) And applications utilizing the characteristics of the antibody. Among its uses are analysis of characteristic structures appearing in the CD44 molecule, analysis of characteristics of tumor cells appearing in tumor tissue, and identification techniques.
The CD44 molecule (including the CD44H molecule and the variable isoform CD44 molecule, an mRNA transcript produced by alternative splicing of the gene), like other transmembrane glycoprotein receptors, is phosphorylated, glycosylated, some species The CD44 isoform has a wide range of post-translational modifications, including glycosaminoglycan chains. The present invention provides a means for elucidating whether or not the function of the CD44 molecule is modified by such modification. In particular, the present invention makes it possible to analyze a biological activity phenomenon observed with a specific modification occurring at a specific site on the CD44 molecule, particularly a specific sugar chain modification of the stem region of CD44, It is possible to provide a technique that can change the motility and morphology of CD44-expressing cells and the cells existing around them, and identify and discriminate cancer cells.

Protein glycosylation is a complex process requiring a variety of glycosyltransferases and glycosylases (Brockhausen et al., Biochim. Biophys Acta, 1473, 67-95, 1999). The type of sugar to be modified and the modification position thereof vary greatly depending on the type of enzyme expressed in the cell and the regulation of the expression level and activity. The importance of glycosylation in vivo has been demonstrated using various glycosyltransferase-deficient mice (Stanley et al., FASEB J., 9, 1436-1444, 1995). However, the target protein and the function of sugar chain modification at a specific position are not fully understood. It is not clear what enzymes are involved in CD44, how glycosylation occurs at specific sites, and how important the modification in the stem region is for its function. In general, protein glycosylation is thought to have a function of stabilizing by limiting access to proteases that act on degradation, and a function of interacting with proteins that have an affinity for a specific sugar. It has been.
In this respect, antibodies having the characteristic immunological reactivity of the present invention, particularly antibodies showing characteristic properties directed to (a specific sequence of) a stem region (common sequence) that appears in common in the CD44 molecule are attracting attention. The In contrast to antibodies obtained by the present invention whose binding is inhibited by the state of sugar chain modification, CD44 of tumor tissue shows different reactivity, for example, it reacts with CD44 expressed in breast cancer and laryngeal cancer. It is an interesting phenomenon that does not react with stromal cells around the tumor.
On the other hand, another antibody obtained in the present invention varies depending on the tumor type, and in breast cancer, it reacts with the CD44 molecule of the tumor and does not react with the stroma. It exhibits the characteristic of showing completely opposite reactivity of not staining.

In the present specification, the term “antibody” may be used in a broad sense, and the desired CD44 molecule, CD44 protein polypeptide and related peptide fragments (of the extracellular domain of CD44 protein). A monoclonal antibody against a site consisting of a continuous amino acid sequence present in the inner extracellular domain, particularly a continuous amino acid sequence present in the stem region of CD44 and a sugar chain-modified peptide fragment thereof (provided that SEQ ID NO: 1 An antibody composition having specificity for a single antigen or a variety of antigenic determinants (epitope), excluding monoclonal antibodies to the continuous 163rd to 186th amino acid sequence sites present in the amino acid sequence represented by Well, including monovalent or multivalent antibodies as well as polyclonal and monoclonal antibodies, In addition, it also represents an intact molecule and fragments and derivatives thereof, including fragments such as F (ab ′) ₂ , Fab ′ and Fab, and further comprising at least two antigen or epitope binding sites. A chimeric antibody or a hybrid antibody, or a bispecific recombinant antibody such as a quadrome or triome, an interspecific hybrid antibody, an anti-idiotype antibody, or a chemically modified or processed antibody. Applying known cell fusion or hybridoma technology and antibody engineering, antibodies obtained using synthetic or semi-synthetic technology, and conventional technologies known from the viewpoint of antibody production Or antibodies prepared using DNA recombination techniques, neutralizing features for target antigenic substances or target epitopes described and defined herein. It may include antibodies that have sex or antibodies that have binding properties. A particularly preferred antibody of the present invention is the amino acid represented by SEQ ID NO: 1 of the stem region of the extracellular domain of the CD44 protein and the region that continues to the region immediately before the transmembrane domain (TM). From the amino acid residue of Thr ¹³⁰ adjacent to the globular region consisting of amino acid residues up to Cys ¹²⁹ on the N-terminal side of the sequence (including the 20 amino acid residues of the signal peptide) Those that can specifically identify the continuous amino acid sequence found in the region up to the 268th amino acid residue Glu (excluding known ones), such as a characteristic reaction to sugar chain modification It is possible to show sex and discriminability, and to recognize them separately. A characteristic sequence of the stem region of the CD44 protein of the present invention, for example, a site consisting of a continuous amino acid sequence present in the stem region has at least 2 to 159 amino acid residues, preferably at least at least amino acid residues. And those specifically binding to or recognizing specifically those of 4 to 50, more specifically, the amino acid sequence represented by SEQ ID NO: 1 of the CD44H protein (signal peptide 20 Pro ²²⁰ -Pro ²³⁰ or Asp ²⁴³ -Gln ²⁵⁰ , the amino acid sequence represented by SEQ ID NO: 1 of CD44 protein (count including 20 amino acid residues of the signal peptide) ) Pro ²⁰⁵ -Ile ²¹⁹ or Thr ¹³⁰ -Arg ¹⁶² or an antibody against a recognition site comprising one or more of the nearby amino acid residues including them.

  Monoclonal antibodies produced against antigenic material are produced using any method that can provide for the production of antibody molecules by a series of cell lines in culture. The modifier “monoclonal” indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. Don't be. Individual monoclonal antibodies are those that contain a population of antibodies that are identical except that only a small amount of naturally occurring variants may be present. Monoclonal antibodies have a high specificity, which is directed against sites with a single antigenicity. In contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different epitopes, each monoclonal antibody is directed against a single antigenic determinant on that antigen. It is what is directed. In addition to its specificity, monoclonal antibodies are also superior in that they are synthesized by hybridoma culture and have no or little contamination with other immunoglobulins. Monoclonal antibodies include hybrid antibodies and recombinant antibodies. They can either replace the variable domain with a constant domain (eg, a humanized antibody) or replace the light chain with a heavy chain, regardless of its origin or type of immunoglobulin class or subclass, as long as it exhibits the desired biological activity. Can be obtained by replacement, replacement of one chain with another, or fusion with a heterogeneous protein (eg, US Pat. No. 4,816,567; Monoclonal Antibody Production Techniques and Applications, pp .79-97, Marcel Dekker, Inc., New York, 1987).

  Examples of suitable methods for producing monoclonal antibodies include the hybridoma method (G. Kohler and C. Milstein, Nature, 256, pp.495-497 (1975)); the human B cell hybridoma method (Kozbor et al., Immunology). Today, 4, pp.72-79 (1983); Kozbor, J. Immunol., 133, pp.3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63, 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)) (human monoclonal antibody (Methods for production); U.S. Pat.No. 4,946,778 (technique for the production of single chain antibodies) and the following literature on antibodies: S. Biocca et al., EMBO J, 9, pp .101-108 (1990); RE Bird et al., Science, 242, pp.423-426 (1988); MA Boss et al., Nucl. Acids Res., 12, pp.3791-3806 (1984); J. Bukovsky et al., Hybridoma, 6, pp.219-228 (1987); M. DAINO et al., Anal. Biochem., 166, pp.223-229 (1987); JS Huston et al., Proc. Natl. Acad. Sci. USA, 85, pp. 5879-5883 (1988); PT Jones et al., Nature , 321, pp.522-525 (1986); JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 121 (Immunochemical Techniques, Part I: Hybridoma Technology and Monoclonal Antibodies), Academic Press, New York (1986); S. Morrison et al., Proc. Natl. Acad. Sci. USA, 81, pp. 6851-6855 (1984); VT Oi et al., BioTechniques, 4, pp. 214-221 (1986) L. Riechmann et al., Nature, 332, pp.323-327 (1988); A. Tramontano et al., Proc. Natl. Acad. Sci. USA, 83, pp.6736-6740 (1986); C Wood et al., Nature, 314, pp.446-449 (1985); Nature, 314, pp.452-454 (1985) or references cited therein (see references for descriptions therein) Are included in the disclosure herein).

As long as they exhibit the desired biological activity, the monoclonal antibodies according to the present invention have a corresponding sequence of an antibody in which part of the heavy and / or light chain is derived from a specific species or belongs to a specific antibody class or subclass. While the same or homologous, the remainder of the chain is a “chimeric” antibody (immunoglobulin) that is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass. In particular (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81, pp. 6851-6855 (1984)).
Hereinafter, 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 is a monoclonal antibody obtained by utilizing cell fusion technology using myeloma cells (for example, G. Kohler and C. Milstein, Nature, 256, pp.495-497 (1975)). An antibody is preferable, and for example, it can be prepared by the following steps.
1. 1. Preparation of immunogenic antigen 2. Immunization of animals with immunogenic antigens 3. Preparation of myeloma cells (myeloma cells) 4. Cell fusion between antibody-producing cells and myeloma cells 5. Selection and monocloning of hybridomas (fusion cells) Production of monoclonal antibodies

1. Preparation of immunogenic antigen Antigens are described in Screaton, GR et al., Proc. Natl. Acad. Sci. USA., 89, pp12160-12164 (1992); Swiss-Prot: accession number P16070 A sequence obtained by expressing a stem region domain fragment of CD44 as a recombinant protein or a fragment derived therefrom can be used by referring to the sequence and applying techniques known in the art.
The antigen can be used to immunize an animal as it is or mixed with an appropriate adjuvant, but may be an immunogenic conjugate or the like. For example, the antigen used as an immunogen is a fragment of the CD44 protein, or a characteristic sequence region selected based on its amino acid sequence, and obtained as a recombinant polypeptide by DNA cloning technology, or a characteristic sequence It may be a synthetic polypeptide fragment obtained by designing and chemically synthesizing a poly or oligopeptide. In addition, the fragment can be combined with various carrier proteins via an appropriate condensing agent to form an immunogenic conjugate such as a hapten-protein, and this can be used to react only with a specific sequence (or only a specific sequence). Can also be used to design monoclonal antibodies. A cysteine residue or the like can be added in advance to the polypeptide to be designed so that an immunogenic conjugate can be easily prepared. In binding to carrier proteins, the carrier proteins can first be activated. For such activation, introduction of an activated linking group can be mentioned.
The activated linking group includes (1) activated ester or activated carboxyl group, such as nitrophenyl ester group, pentafluorophenyl ester group, 1-benzotriazole ester group, N-succinimide ester group, etc. A dithio group, such as a 2-pyridyldithio group, can be mentioned. Examples of carrier proteins include keyholes, limpets, hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, globulins, polylysine and other polypeptides, and bacterial cell components such as BCG.

2. Immunization of animals with immunogenic antigens Immunization can be performed by methods known to those skilled in the art. For example, Shigeru Muramatsu, et al., Laboratory Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985, Japan Biochemistry Society, Secondary Biochemistry Experiment Course 5, Immunobiochemistry Research Method, Tokyo Chemical Doujin, 1986, Japanese Biochemical Society, New Chemistry Experiment Course 12, Molecular Immunology III, Antigen / Antibody / Complement, Tokyo Chemical Doujin Can be carried out according to the method described in 1992 and the like. Immunization is accomplished by injecting the immunizing agent (optionally with an adjuvant) into the mammal one or more times. 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 antigen protein (peptide) or a related peptide fragment thereof. The immunizing agent may be used in the form of a conjugate with a protein known to be immunogenic in the mammal to be immunized (for example, the above carrier proteins). Examples of adjuvants include Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG, lipid A, liposome, aluminum hydroxide, silica and the like. Immunization is performed using, for example, mice such as BALB / c, hamsters, and other suitable animals. The dose of the antigen is, for example, about 1 to about 400 μg / animal for mice, generally injected into the peritoneal cavity or subcutaneously of the host animal, and thereafter every 1 to 4 weeks, preferably every 1 to 2 weeks. Repeat booster immunization 2-10 times intraperitoneally, subcutaneously, intravenously or intramuscularly. As immunization mice, BALB / c mice, BA1 / c mice and other mice F1 mice, and the like can be used. If necessary, an antibody titer measurement 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 immunized animal thus obtained and includes, for example, antiserum, polyclonal antibody and the like.

3. Preparation of myeloma cells (myeloma cells) Infinitely proliferative strains (tumor cell lines) used for cell fusion can be selected from cell lines that do not produce immunoglobulins and should be selected from those known in the art. For example, P3-NS-1-Ag4-1 (NS-1, Eur. J. Immunol., 6: 511-519, 1976), SP-2 / 0-Ag14 (SP-2, Nature, 276: 269-270, 1978), P3-X63-Ag8-U1 derived from mouse myeloma MOPC-21 cell line (P3U1, Curr. Topics Microbiol. 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. 8-Azaguanine-resistant mouse myeloma cell line is prepared by adding antibiotics such as penicillin and amikacin, fetal calf serum (FCS), etc. to cell culture medium such as Dulbecco's MEM medium (DMEM medium) and RPMI-1640 medium. It is subcultured in a medium supplemented with azaguanine (for example, 5-45 μg / ml), but it can be subcultured in a normal medium 2 to 5 days before cell fusion to prepare the required number of cell lines. The cell line used was prepared by thawing the cryopreserved strain completely at about 37 ° C, washing it with a normal medium such as RPMI-1640 medium three times or more, and then culturing in the normal medium to prepare the required number of cell lines. There may be.

4). Cell fusion between antibody-producing cells and myeloma cells [2. After immunization, the spleen is removed 2 to 5 days after the final immunization, and a spleen cell suspension is obtained from the animal immunized according to the step of immunization of animal with immunogenic antigen. In addition to spleen cells, lymph node cells in various parts of the body can be obtained and used for cell fusion. The spleen cell suspension thus obtained and 3. The myeloma cell line obtained according to the above step is placed in a cell medium such as minimum essential medium (MEM medium), DMEM medium, RPMI-1640 medium, etc., and a cell fusion agent such as polyethylene glycol (PEG) is added. As the cell fusion agent, those known in various other fields can be used, and examples thereof include inactivated Sendai virus (HVJ: Hemagglutinating Virus of Japan). Preferably, for example, 0.5 to 2 ml of 30 to 60% polyethylene glycol can be added, polyethylene glycol having a molecular weight of 1,000 to 8,000 can be used, and polyethylene glycol having a molecular weight of 1,000 to 4,000 can be more preferably used. The concentration of polyethylene glycol in the fusion medium is preferably 30 to 60%, for example. If necessary, for example, 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 the fusion is, for example, 1: 1 to 20: 1, more preferably 4: 1 to 7: 1.
The fusion reaction is performed for 1-10 minutes and then a cell culture medium such as RPMI-1640 medium is added. The fusion reaction treatment can be performed multiple times. After the fusion reaction treatment, the cells are separated by centrifugation or the like and then transferred to a selection medium.

5). Selection and Monocloning of Hybridoma (Fusion Cell) Examples of the selection medium include media such as hypoxanthine, aminopterin and thymidine, such as FCS-containing MEM medium and RPMI-1640 medium, so-called HAT medium. In general, the selective medium can be replaced by adding a volume equal to the volume dispensed to the culture plate the next day, and then replacing the HAT medium half by half every 1 to 3 days. You can also change this. On the 8th to 16th day after the fusion, the medium can be changed every 1 to 4 days with a so-called HT medium excluding aminopterin. For example, mouse thymocytes can be used as a feeder, which may be preferred.
The culture supernatant of the culture well in which the hybridoma grows can be measured using a measurement system such as radioimmunoassay (RIA), enzyme immunoassay (ELISA) or fluorescence immunoassay (FIA), or a fluorescence-induced cell separator (FACS). Then, screening is performed by using a predetermined fragment peptide as an antigen, or by measuring a target antibody using a labeled anti-mouse antibody.
The hybridoma producing the target antibody is cloned. Cloning can be done by picking up colonies in agar or by limiting dilution. More preferably, the limiting dilution method can be used. Cloning is preferably performed multiple times.

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

In addition, transgenic mice or other organisms such as other mammals can be used to express antibodies (including humanized antibodies) to the immunogenic protein (polypeptide) product of the present invention. it can.
It is also possible to determine the sequence of the antibody thus obtained in large quantities, or to produce an antibody by a gene recombination technique using a nucleic acid sequence encoding the antibody obtained from a hybridoma strain. The nucleic acid encoding the monoclonal antibody can be isolated and sequenced by conventional techniques such as using an oligonucleotide probe that can specifically bind to the gene encoding the heavy or light chain of the mouse antibody. . Once isolated, the DNA can be put into a suitable expression vector and put into a host cell such as CHO or COS. The DNA can be modified, for example, by substituting a sequence encoding a human heavy chain or light chain constant region domain in place of the homogeneous mouse sequence (Morrison et al., Proc Natl. Acad. Sci. USA, 81: 6581, 1984). Thus, it is possible to prepare a chimeric antibody or a hybrid antibody having a desired binding specificity. The antibody can also be modified by applying a chemical protein synthesis technique including the use of a condensing agent as described below to prepare a chimeric antibody or a hybrid antibody.
Humanized antibodies can be performed by techniques known in the art (eg, Jones et al., Nature, 321: pp. 522-525 (1986); Riechmann et al., Nature, 332: pp .323-327 (1988); Verhoeyen et al., Science, 239: pp.1534-1536 (1988)). Human monoclonal antibodies can also be performed 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. 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York (1987)). Methods for producing bispecific antibodies are also known in the art (Millstein et al., Nature, 305: pp. 537-539 (1983); WO93 / 08829; Traunecker et al., EMBO J., 10: pp.3655-3659 (1991); Suresh et al., "Methods in Enzymology", Vol. 121, pp.210 (1986)).
Further, these antibodies may be used in the form of antibody fragments such as Fab, Fab ′, and F (ab ′) ₂ obtained by treating with an enzyme such as trypsin, papain, or pepsin and optionally reducing them.

The antibodies can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).
Any method known in the art can be used to conjugate each antibody to a detectable atomic group, such as David et al., Biochemistry, 13, 1014-1021 (1974); Pain et al, J. Immunol. Meth., 40: pp. 219-231 (1981); and “Methods in Enzymology”, Vol. 184, pp. 138-163 (1990). As an antibody for imparting a label, an IgG fraction, or a specific binding part Fab ′ obtained by reduction after digestion with pepsin can be used. Examples of the label in these cases include enzymes (peroxidase, alkaline phosphatase, β-D-galactosidase, etc.), chemical substances, fluorescent substances, and radioisotopes as described below.

  Detection and measurement in the present invention can be performed by immunostaining such as tissue or cell staining, immunoassay such as competitive immunoassay or non-competitive immunoassay, radioimmunoassay (RIA), enzyme immunoassay (EIA), Photoimmunoassay (FIA), fluorescent antibody method (FAT), chemiluminescence immunoassay (CLIA), chemiluminescence / enzyme immunoassay (CLEIA), fluorescence polarization immunoassay (FPIA), flow cytometry ( FACS) or the like can be used, and the BF separation may or may not be performed. RIA, ELISA, FIA and the like are preferable, and a sandwich type assay is also included. For example, in a sandwich type assay, one is an antibody to the CD44 protein and its related peptide fragment, the other is another type of antibody to the CD44 protein, and one is detectably labeled. Other antibodies capable of recognizing the same antigen are immobilized on the solid phase. Incubation treatment is performed in order to sequentially react the sample, labeled antibody, and solid-phased antibody as necessary. After separating the unbound antibody, the labeled product is measured. The amount of label measured is proportional to the amount of antigen, ie, antigen having a recognition site on the CD44 molecule. This assay is called a simultaneous sandwich type assay, a forward sandwich type assay, a reverse sandwich type assay or the like depending on the order of addition of the insolubilized antibody or the labeled antibody. For example, washing, stirring, shaking, filtration, or antigen pre-extraction are appropriately employed in the measurement process under specific circumstances. Other measurement conditions such as the concentration of a particular reagent, buffer, etc., temperature or incubation time can be varied according to factors such as the concentration of antigen in the sample and the nature of the sample. A person skilled in the art can perform measurement by appropriately selecting optimum conditions effective for each measurement using a normal experimental method.

Many carriers capable of immobilizing an antigen or antibody are known, and in the present invention, they can be appropriately selected and used. Various carriers used for antigen-antibody reactions and the like are known, and of course, in the present invention, these carriers can be selected and used. Particularly preferably used are, for example, glass, such as activated glass, porous glass, silica gel, silica-alumina, alumina, magnetized iron, magnetized alloy and other inorganic materials, polyethylene, polypropylene, polyvinyl chloride, polyfluoride Cross-linked albumin such as vinylidene, polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, cross-linked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer Natural or modified cellulose such as collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethylcellulose, cellulose acetate, Organic polymer materials such as polyamides such as dextran and nylon, polyurethane and polyepoxy resins, and those obtained by emulsion polymerization of them, cells, erythrocytes, etc. If necessary, functional groups with silane coupling agents etc. The thing which introduce | transduced is mentioned. In addition, the carrier may be provided with an identifiable label.
In addition, filter paper, beads, inner walls of test containers, eg cells made of synthetic materials such as test tubes, cuvettes, titer plates, titer wells, glass cells, synthetic resin cells, fine particles, micro particles, nanoparticles, glass rods, synthetic Examples include rods made of materials, rods with thickened or thinned ends, rods with rounded or flattened ends, and solid material (object) surfaces such as thin plates. .

An antibody can be bound to these carriers, and preferably a monoclonal antibody that specifically reacts with the antigen obtained in the present invention can be bound. The binding between the carrier and those involved in the antigen-antibody reaction may be performed by a physical method such as adsorption, a chemical method using a condensing agent, an activated one, etc. It can be performed by a method using a chemical bonding reaction.
Labels include enzymes, enzyme substrates, enzyme inhibitors, prosthetic molecules, coenzymes, enzyme precursors, apoenzymes, fluorescent materials, dye materials, chemiluminescent compounds, luminescent materials, chromogenic materials, magnetic materials, metal particles such as gold Examples thereof include radioactive materials such as colloids. Examples of the enzyme include oxidoreductases such as dehydrogenase, reductase, and oxidase, such as transferases that catalyze transfer of amino groups, carboxyl groups, methyl groups, acyl groups, phosphate groups, and the like, such as ester bonds, Examples include hydrolase, lyase, isomerase, ligase and the like that hydrolyze glycoside bonds, ether bonds, peptide bonds and the like. The enzyme can be used for detection by using a plurality of enzymes in combination. For example, enzymatic cycling can be used.
Typical radioactive isotope elements for labeling are [aUP], [ ¹² iI], [ ¹³ OI],
[aH], [ ¹⁴ C], [aiS] and the like.

Typical enzyme labels include peroxidase such as horseradish peroxidase (HRP), galactosidase such as E. coli β-D-galactosidase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, acetylcholinesterase, Examples thereof include alkaline phosphatase such as catalase, bovine small intestine alkaline phosphatase, and Escherichia coli alkaline phosphatase.
When alkaline phosphatase is used, substrates such as umbelliferone derivatives such as 4-methylumbelliferyl phosphate, phosphorylated phenol derivatives such as nitrophenyl phosphate, enzymatic cycling systems using NADP, luciferin derivatives, dioxetane derivatives, etc. It can be measured by the fluorescence, luminescence, etc. that occur when used. Luciferin and luciferase systems can also be used. When catalase is used, it reacts with hydrogen peroxide to produce 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 hardly 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 may use a plurality of different types of labels. In such cases, it may also be possible to perform multiple measurements continuously or discontinuously and simultaneously or separately.
In the present invention, 4-hydroxyphenylacetic acid, o-phenylenediamine (OPD), tetramethylbenzidine (TMB), 5-aminosalicylic acid, 3,3-diaminobenzidine tetrahydrochloride (DAB), 3-amino -9-ethylcarbazole (AEC), tyramine, luminol, lucigenin, luciferin and their derivatives, HRP such as horseradish peroxidase, lumigen PPD, (4-methyl) umbelliferyl-phosphate, p-nitrophenol-phosphorus Umbelliferyl galactosides such as acid, phenol-phosphate, bromochloroindolyl phosphate (BCIP), AMPAK ^TM (DAKO), AmpliQ ^TM (DAKO) and alkaline phosphatase, 4-methylumbelliferyl-β-D-galactoside Nitrophenyl galactoside such as o-nitrophenol-β-D-galactoside and β-D-galactosidase, Combinations of glucose-6-phosphate dehydrogenase, ABTS, etc. and enzyme reagents such as glucose oxidase can also be used, quinol compounds such as hydroquinone, hydroxybenzoquinone, hydroxyanthraquinone, thiol compounds such as lipoic acid and glutathione, phenol derivatives, ferrocene What can form a derivative etc. by the action of an enzyme etc. can be used.
Examples of fluorescent substances or chemiluminescent compounds include fluorescein isothiocyanate (FITC), for example, rhodamine derivatives such as rhodamine B isothiocyanate, tetramethylrhodamine isothiocyanate (RITC), tetramethylrhodamine isothiocyanate isomer R (TRITC), 7-amino- Examples include 4-coumarin-3-acetic acid, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, acridinium salts, luminiferin, luciferase, equolin, and other luminols, imidazoles, oxalates, rare earth chelates, and coumarin derivatives. It is done. As the fluorescent dye, those used in the usual fluorescent antibody method can be used. Detection of signals generated including color development, fluorescence, and the like can be performed visually, but a known device can also be used. For example, a fluorescent photometer, a plate reader, or the like can be used. Further, in order to detect a signal emitted from a radioisotope (isotope) or the like, a known apparatus can be used, for example, a gamma counter, a scintillation, or the like can be used.

The labeling can be performed using 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., and can be easily performed by a known method or those skilled in the art. It can be applied by appropriately selecting from the methods that can be carried out, and methods modified by them. In addition, 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 bissuccinimidyl succinate. , Bisdiazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxy (SMCC), N-sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl) aminobenzoate, N-succinimidyl 4- (1-maleimidophenyl) Butyrate, N- (ε-maleimidocaproyloxy ) Succinimide (EMCS), iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4'-dithiopyridyl) propionimidate, methyl-4-mercaptobutyrylimidate, methyl-3-mercaptopropion Examples include imidate and N-succinimidyl-S-acetylmercaptoacetate.

According to the measurement method of the present invention, a labeled antibody reagent such as a monoclonal antibody in which a substance to be measured is labeled with an enzyme and an antibody bound to a carrier can be reacted sequentially or simultaneously. . The order in which reagents are added depends on the type of carrier system selected. When using beads such as sensitized plastics, first place a labeled antibody reagent such as a monoclonal antibody labeled with an enzyme together with a specimen sample containing the substance to be measured in an appropriate test tube, and then Measurements can be made by adding beads such as sensitized plastic.
In the quantification method of the present invention, an immunological measurement method is used. In this case, as a solid phase carrier, polystyrene, polycarbonate, polypropylene or polyvinyl balls, microplates that adsorb proteins such as antibodies well are used. Various materials and forms such as sticks, fine particles or test tubes can be arbitrarily selected and used.
The measurement can be performed in an appropriate buffer system so as to maintain an optimum pH, for example, a pH of about 4 to about 9. Particularly suitable buffers include, for example, acetate buffer, citrate buffer, phosphate buffer, Tris buffer, triethanolamine buffer, borate buffer, glycine buffer, carbonate buffer, Tris-HCl A buffering agent etc. are mentioned. The buffering agents can be mixed and used at any ratio. The antigen-antibody reaction is preferably performed at a temperature between about 0 to about 60 ° C.
Incubation treatment of antibody reagents such as monoclonal antibodies labeled with enzymes, antibody reagents bound to carriers, and substances to be measured can be performed until equilibrium is reached, but the equilibrium of the antigen-antibody reaction has been achieved. The reaction can be stopped after a limited incubation by separating the solid and liquid phases 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. Can be measured. The measurement operation can be performed using an automated measuring device, and a luminescence detector, a photo detector or the like is used to detect and measure a display signal generated by converting the substrate by the action of an enzyme. You can also

In the antigen-antibody reaction, appropriate measures can be taken to stabilize the reagents used, substances to be measured, and labels such as enzymes, or to stabilize the antigen-antibody reaction itself. In addition, proteins, stabilizers, surfactants, chelating agents, etc. can be added to the incubation solution to eliminate non-specific reactions, reduce the inhibitory effect, or activate the measurement reaction. It can also be added. As the chelating agent, ethylenediaminetetraacetate (EDTA) is more preferable.
A blocking treatment for preventing a non-specific binding reaction commonly employed in the field or known to those skilled in the art may be performed, for example, normal serum proteins such as mammals, albumin, skim milk, milk fermentation substances , Collagen, gelatin and the like. As long as the purpose is to prevent non-specific binding reaction, those methods are not particularly limited and can be used.
As a sample to be measured by the measurement method of the present invention, any form of solution, colloidal solution, non-fluid sample, etc. can be used, but preferably a sample derived from a living organism such as thymus, testis, intestine, kidney, brain, breast cancer. , Ovarian cancer, colorectal cancer, blood, serum, plasma, joint fluid, cerebrospinal fluid, pancreatic fluid, bile fluid, saliva, amniotic fluid, urine, other body fluids, cell culture fluid, tissue culture fluid, tissue phomodulate, biopsy Samples, tissues, cells and the like can be mentioned.
When various analysis / quantification methods including these individual immunological measurement methods are applied to the measurement method of the present invention, special conditions and operations are not required to be set. A measurement system related to the target substance of the present invention or a substance having substantially the same activity may be constructed by adding ordinary technical considerations to those skilled in the art to the normal conditions and operation methods in each method. .

  For details of these general technical means, refer to reviews, books, etc. [For example, Hiroshi Irie, “Radioimmunoassay”, Kodansha, published in 1974; Hiroshi Irie, “Second radioimmunoassay” "Kodansha, published in 1979; edited by Eiji Ishikawa et al.," Enzyme Immunoassay ", Medical School, published in 1978; edited by Eiji Ishikawa et al.," Enzyme Immunoassay "(2nd edition), Medical School, Showa Published in 1957; edited by Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition), Medical School, published in 1987; HV Vunakis et al. (Ed.), “Methods in Enzymology”, Vol. 70 (Immunochemical Techniques, Part A), Academic Press, New York (1980); JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 73 (Immunochemical Techniques, Part B), Academic Press, New York (1981) JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 74 (Immunochemical Techniques, Part C), Academic Press, New York (1981); JJ La ngone et al. (ed.), "Methods in Enzymology", Vol. 84 (Immunochemical Techniques, Part D: Selected Immunoassays), Academic Press, New York (1982); JJ Langone et al. (ed.), "Methods in Enzymology ", Vol. 92 (Immunochemical Techniques, Part E: Monoclonal Antibodies and General Immunoassay Methods), Academic Press, New York (1983); JJ Langone et al. (ed.)," Methods in Enzymology ", Vol. 121 (Immunochemical Techniques, Part I: Hybridoma Technology and Monoclonal Antibodies), Academic Press, New York (1986); JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 178 (Antibodies, Antigens, and Molecular Mimicry ), Academic Press, New York (1989); M. Wilchek et al. (Ed.), "Methods in Enzymology", Vol. 184 (Avidin-Biotin Technology), Academic Press, New York (1990); JJ Langone et al. (ed.), "Methods in Enzymology", Vol. 203 (Molecular Design and Modeling: Concepts and Applications, Part B: Anibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs), Academic Press, New York (199 1) etc. or references cited therein (the description contained therein is hereby incorporated by reference into the disclosure)].

One embodiment of the use of the antibody of the present invention is a method for detecting binding between the anti-CD44 antibody and a target CD44 molecule in a liquid phase system. For example, the labeled antibody is brought into contact with the biological sample to bind the labeled antibody and the target CD44 molecule, and the conjugate is separated. Separation can be performed by a method of separating a conjugate of target CD44 molecule + labeled antibody by a known separation means (chromatography method, solid phase method, etc.). A method according to a known Western blot method can also be employed. When an enzyme is used as the label, the label signal is measured by adding a substrate that develops color by enzymatic action and optically measuring the amount of degradation of the substrate to determine the enzyme activity. The amount of antibody is calculated by conversion and comparison with the standard value. When using a radioactive isotope, the radiation dose emitted by the radioactive isotope is measured with a scintillation counter or the like. In addition, when a fluorescent dye is used, the amount of fluorescence may be measured by a measuring device combined with a fluorescence microscope.
Another method in the liquid phase system is to bring the antibody (primary antibody) into contact with the biological sample to bind the primary antibody and the target CD44 molecule, and label the predetermined antibody (secondary antibody) labeled with this conjugate. Once bound, the label signal in the tripartite conjugate is detected. Alternatively, in order to further enhance the signal, an unlabeled secondary antibody may be first bound to an antibody + antigen protein conjugate, and a labeling substance may be bound to this secondary antibody. Such binding of the labeling substance to the secondary antibody can be performed, for example, by biotinylating the secondary antibody and avidinizing the labeling substance. Alternatively, an antibody (tertiary antibody) that recognizes a partial region (for example, Fc region) of the secondary antibody may be labeled and the tertiary antibody may be bound to the secondary antibody. The primary antibody and the secondary antibody may both be monoclonal antibodies, or one of the primary antibody and the secondary antibody may be a polyclonal antibody. Separation of the conjugate from the liquid phase and detection of the signal can be performed as described above.

Another form of use of the antibody obtained in the present invention is a method of testing the binding between the antibody and the target CD44 molecule in a solid phase system. This method in the solid phase system is a preferable method for detecting a very small amount of target CD44 molecule and simplifying the operation. That is, in this solid phase system, the antibody of the present invention is immobilized on a resin plate or membrane, the target CD44 molecule is bound to this immobilized antibody, unbound proteins are washed away, and then the antibody remaining on the plate + This is a method of detecting the signal of the labeled antibody by binding the required labeled antibody to the target CD44 molecule conjugate. This method is a so-called “sandwich method”. When an enzyme is used as a marker, this method is widely used as an “ELISA (enzyme linked immunosorbent assay)”. Both of the two types of antibodies can be monoclonal antibodies, or one of them can be a polyclonal antibody.
Examples of the CD44 molecule measurement system in the present invention include immunostaining for tissues, protein measurement systems such as immunoelectron microscope, expression gene measurement systems such as in situ hybridization combined with immunostaining, and tissue extraction. EIA, RIA, FIA, LIA, Western blotting and other protein measurement systems, Northern blotting, Southern blotting, dot blot, RNase protection assay, RT-PCR (reverse transcription polymerase chain reaction), Real-Time PCR, Protein detection systems such as EIA, RIA, FIA, LIA, and Western blotting can be advantageously used for a combination of an expression gene measurement system such as competitive PCR and a detection system using antibodies, and for blood and body fluids.
In the EIA measurement system, for example, in the competition method, an anti-CD44 antibody is used as a solid-phased antibody, and a labeled antigen and an unlabeled antigen (antigens include CD44 or a fragment peptide thereof) are used. In the non-competitive method, for example, the sandwich method, an immobilized anti-CD44 antibody or a labeled anti-CD44 antibody can be used. In addition, the anti-CD44 antibody can be directly labeled, or an antibody against the anti-CD44 antibody can be labeled without being immobilized. Alternatively, it can be carried out in a solid phase. As a sensitivity amplification method, for example, in combination with a non-enzyme labeled primary antibody, a method using a polymer, an enzyme and a primary antibody (envision reagent applied; Enhanced polymer one-step staining (EPOS)) is mentioned. In combination with a non-enzyme labeled secondary antibody, for example, a combination of an enzyme such as PAP (peroxidase-antiperoxidase) and an anti-enzyme antibody complex, and a biotin-labeled secondary antibody such as SABC (avidin-biotinylated peroxidase complex) Biotin-labeled enzyme-avidin complex combination, ABC (streptavidin-biotin complex) method, LSAB (labeled streptavidin-biotin) method and other biotin-labeled secondary antibodies and biotin-labeled enzyme-streptavidin complex combinations, A combination of SABC, biotin-labeled tyramide, and enzyme-labeled streptavidin, such as the amplification method, and polymers labeled with secondary antibodies and enzymes. I can get lost.

Both the immobilized anti-CD44 antibody of the present invention and the anti-CD44 antibody labeled with a detectable label are useful for quantitatively measuring the molecular weight of CD44 having a specific characteristic structural property or form. It is.
In the present invention, at least the following steps:
At least the following steps:
(a) contacting the test biological sample with a carrier on which the anti-CD44 antibody according to any one of claims 1 to 17 is immobilized;
(b) washing the solid phase carrier brought into contact with the biological sample;
(c) a step of contacting an anti-CD44 antibody labeled with a solid phase carrier contacted with the biological sample and recognizing an epitope different from the immobilized anti-CD44 antibody;
(d) measuring a solid-phased label or a label that is free; and
(e) The labeled amount measured in step (d) is used as an index of CD44 amount,
(1) Detecting the difference in the sugar chain of CD44 expressed on cells,
(2) detecting differences in cell glycosylation in tumor tissue, and
(3) Distinguishing between tumor cells and non-tumor cells in a tumor tissue A method for measuring a biological activity phenomenon associated with CD44 is provided, comprising the step of selecting from the group consisting of:
Another embodiment of the measurement method is the same as in the step (a) using a carrier on which the anti-CD44 antibody of (c) is immobilized instead of the anti-CD44 antibody according to any one of claims 1 to 17. On the other hand, using the antibody labeled with the anti-CD44 antibody according to any one of claims 1 to 17 instead of the anti-CD44 antibody of (c) above, the same treatment as in the above step (c) If you want to do.
In this method, the target CD44 protein abundance is preferably measured quantitatively.

In the present invention, at least the following steps:
(a) subjecting the test biological sample to tissue immobilization treatment;
(b) a step of using the tissue-fixed specimen prepared in step (a) as a section;
(c) subjecting the sectioned tissue to immunohistochemical staining with the anti-CD44 antibody according to any one of claims 1 to 17;
(d) Using the degree of immunohistochemical staining in the test biological sample as an index,
(1) Detecting the difference in the sugar chain of CD44 expressed on cells,
(2) detecting differences in cell glycosylation in tumor tissue, and
(3) Distinguishing between tumor cells and non-tumor cells in a tumor tissue. A method for measuring a biological activity phenomenon associated with CD44 is provided. By performing quantitative measurement here, tumor cells and non-tumor cells can be distinguished.
The antibody obtained in the present invention can be used to construct a diagnostic kit, which is a reagent kit for performing the above method. Since such a kit is commercially available depending on the type of test component, it can be used as the diagnostic kit of the present invention. Except for the use of the antibody and / or labeled antibody provided by the present invention, it can be constituted by each element used in a publicly known kit.
In the present specification, the “target CD44 molecule” may include a CD44 fragment generated by cleavage with a protease in the stem region of the CD44. Further, the target CD44 molecule may include those specifically recognized by the anti-CD44 antibody newly established in the present invention.
Another means for measuring the target CD44 molecule using the antibody obtained in the present invention is a method using a protein microarray (protein chip). As a method for producing a microarray, a method in which a predetermined antibody is immobilized on the surface of a solid support is known. For arraying, technologies similar to those used to create DNA microarrays (DNA chips) can be applied, such as the use of protecting groups that are selectively removed by light irradiation, photolithography technology and solid phase synthesis technology used in semiconductor manufacturing. And a method of performing selective modification in a predetermined region of a minute matrix can be used. For arraying, see, for example, Blawas, AS, et al., Biomaterials, 19, pp595-609, 1998. In a typical procedure, the glass surface is silanized or esterified, coated with avidin (eg, NeutrAvidin ^™ ), and then the biotinylated antibody is plotted and spotted.
In general, the predetermined antibody is covalently bonded to a surface-treated solid support through a spacer or a crosslinker as necessary. A fine piece of polyacrylamide gel may be aligned on the glass surface, and proteins may be bound thereto. In addition, even if an array of microelectrodes is prepared on a silica microarray, an agarose permeation layer containing streptavidin is provided on the electrode as a reaction site, and this site is positively charged to immobilize biotinylated protein. Good. The production of a protein chip can be performed by applying techniques and techniques known to those skilled in the art in this field.
The binding between the antibody molecule and the target CD44 molecule may be detected by various methods such as using a fluorescent substance label, utilizing a surface plasmon resonance phenomenon, or utilizing a crystal oscillator. The present technology may include a program, a data processing system, and an analysis system optimized for processing data such as obtained signals.

As a result of the establishment of the antibody of the present invention, it has been found that CD44 of tumor tissue shows a difference in reactivity with antibodies whose binding is inhibited by the state of sugar chain modification, for example, antibodies 268-1F5 and 294-6F2. It was issued. In particular, antibody 268-1F5 reacts with CD44 expressed in two breast cancers and three laryngeal cancers, but does not react with stromal cells around the tumor, whereas antibody 294-6F2 reacts with it. Sex varies depending on the tumor type: Breast cancer responds to tumor CD44 molecules, does not react to stroma, laryngeal cancer stains stromal cells and does not stain tumor cells. Successfully found to show.
Thus, it is considered that the sugar chain modification affecting the reactivity of antibodies 268-1F5 and 294-6F2 is under different control between tumor cells and stromal cells and also in the type of tumor. It is also possible that CD44, which has a tumor-specific modification, has a special function involved in the malignancy of tumor cells. The results of the present invention probably indicate that the CD44 molecule undergoes site-specific O-linked glycosylation depending on the cell type. These antibodies are involved in glycosylation of CD44 in order to monitor the glycosylation status of these specific sites in vivo, to study their function and regulation mechanism It is expected to be useful for clarifying glycosyltransferase / addition enzymes. For the first time in the present invention, by using an antibody having very characteristic properties, it was possible to clarify the site where the binding to the antibody is inhibited by O-linked sugar chain modification. We succeeded in finding that tumor cells can be identified by the difference.

CD44 is also known to be cleaved by the protease in the stem region and released into the bloodstream (shedding). This shedding is thought to control the ability of cells to adhere to HA and alter signaling through CD44. A variety of cell surface proteases are involved in this processing (Okamoto et al., Oncogene, 18, 1435-1446, 1999), suggesting that MT1-MMP is one such protease. (Kajita et al., J. Cell Biol., 153 :, 893-904., 2001). No other protease candidates have been identified yet, but if different proteases cleave CD44, the cleavage site will vary depending on the respective protease. However, the molecular weight of the cleavage fragment does not provide clear information about the cleavage site. In other words, since the molecular weight of CD44 varies greatly depending on the sugar chain modification, it is dangerous to judge only by molecular weight. In the present invention, a characteristic sequence in the stem region of CD44 is obtained as an epitope of the antibody, and it will be useful for analyzing CD44 fragments produced by different proteases. In addition, depending on the position of the sugar chain modification, the binding of protease to CD44 may be inhibited or promoted. Furthermore, there is a possibility that the way of sugar chain modification is changed by the interaction of protease. Malignant cells show increased CD44 cleavage (Okamoto et al., Am. J. Pathol, 160, 441-447, 2002), and MT1-MMP is also expressed in such tumor tissues (Seiki et al. , Cancer Lett, 194, 1-11, 2003). Thus, the antibodies of the present invention are useful for detecting CD44 fragments cleaved by specific enzymes, such as MT1-MMP, while at the same time being able to reveal their interactions. A sandwich ELISA or the like can be used as the means. This time, a two-step enzyme immunoassay was examined using the two antibodies of the present invention. The result reflected the result by Western blotting. Since it can be quantified by this method, the increase or decrease in the expression level can also be observed. Until now, there have been several reports using the same EIA method, but since the antibody affected by glycosylation was not used, new possibilities were found for the sugar chain function and regulatory mechanism of the CD44 molecule. Can be expected.
In short, CD44 is a cell adhesion molecule that acts as a major receptor for hyaluronic acid and has many biological and disease states such as lymphocyte homing, T cell activation, wound healing, angiogenesis and metastatic spread of tumor cells. Involved in the physical process. However, detailed analysis was not possible due to the complexity of the molecule, such as alternative splicing isomers, various glycosylation modifications and cleavage by different proteases. However, in the present invention, four types of monoclonal antibodies (285-2F12, 284-23F1, 268-1F5 and 294-6F2) that recognize the defined sequence of the CD44 stem region and one type of polyclonal antibody are prepared. In Western blotting, two monoclonal antibodies (268-1F5 and 294-6F2) cannot recognize CD44 expressed in five of the seven tumor cell lines, whereas neuraminidase and O-glycosidase are combined. These antibodies reacted with CD44, which had undergone the above treatment and amino acid substitution mutation at the site where a sugar chain could be added. This indicates that these reactivities are affected by O-linked glycosylation, possibly modification near the recognition site, and the CD44 glycosylation that affects these reactivities includes two breast cancers and three It was found that the tumor cells and stromal cells in the laryngeal cancer tissue are under different control. Antibody 268-1F5 reacted to the tumor cells but not the surrounding stromal cells. On the other hand, the reactivity of antibody 294-6F2 was different between the two tumor types. Thus, these antibodies are useful for detecting and analyzing site-specific glycosylation of CD44, particularly a role not yet known in tumors.

In the specification and drawings, the terms are based on the meanings of terms used by the IUPAC-IUB Commission on Biochemical Nomenclature or conventionally used in the field. The meaning of typical terms is shown below.
Regarding the amino acid sequence:
A: Alanine (Ala) M: Methionine (Met)
C: Cysteine (Cys) N: Asparagine (Asn)
D: Aspartic acid (Asp) P: Proline (Pro)
E: Glutamic acid (Glu) Q: Glutamine (Gln)
F: Phenylalanine (Phe) R: Arginine (Arg)
G: Glycine (Gly) S: Serine (Ser)
H: Histidine (His) T: Threonine (Thr)
I: Isoleucine (Ile) V: Valine (Val)
K: Lysine (Lys) W: Tryptophan (Trp)
L: Leucine (Leu) Y: Tyrosine (Tyr)
For nucleotide sequences:
A, a: Adenine G, g: Guanine
C, c: cytosine T, t: thymine

The present invention will be described in detail with reference to the following examples, which are provided merely for the purpose of illustrating the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application. In the present invention, it should be understood that various embodiments based on the idea of the present specification are possible.
All examples were performed or can be performed using standard techniques, except as otherwise described in detail, and are well known and routine to those skilled in the art. .
Molecular biological techniques including commonly used DNA cloning in the following examples include standard laboratory manuals such as J. Sambrook, EF Fritsch & T. Maniatis, Molecular Cloning: A Laboratory Manual (2nd Edition). , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) & J. Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001) Can be implemented as described. Especially in PCR, R. Saiki et al., Science, 230: 1350, 1985; R. Saiki et al., Science, 239: 487, 1988; HA Erlich (ed.), PCR Technology, Stockton Press, 1989 DM Glover et al. (Ed.), "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press, Oxford University Press (1995); MA Innis et al. (Ed.) , "PCR Protocols: a guide to methods and applications", Academic Press, New York (1990)); MJ McPherson, P. Quirke and GR Taylor (ed.), PCR: a practical approach, IRL Press, Oxford (1991) MA Frohman et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002 (1988), etc., or when using commercially available reagents or kits They use attached protocols (protocols) and attached chemicals.

[Preparation of anti-CD44 monoclonal antibody]
a) Preparation of immunogen
It was prepared by the method of Kajita et al. Described in J. Cell Biol., 153, 893-904, 2001. CDNA (rCD44HS) encoding CD44H stem region (Thr ¹³⁰ -Glu ²⁶⁸ (all based on the amino acid sequence represented by SEQ ID NO: 1)) with FLAG tag at the N-terminus and His ⁶ tag at the C-terminus Was expressed using a bacterial vector, pET3a (Stratagene, La Jolla, Calif.) (FIG. 1). E. coli strain BL21 (DE3) pLysS was transformed with the plasmid and protein expression was induced with 0.4 mM IPTG. Cells were collected and sonicated with a TNC buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl ₂ , 0.02% NaN ₃ ) containing 2 mM PMSF. The supernatant was collected and the His ^6- tag protein was purified on a gel filtration column using chelating Sepharose and AKTA explorer 10S system (Amersham Pharmecia Biotech, Buckinghamshire, UK). The CD44H stem region fragment (GST-tagged RS1 (Thr ¹³⁰ -Arg ¹⁶² )) was also expressed in E. coli and purified in the same manner as rCD44HS. A P1 oligopeptide containing DGHSHGSQEGGA (amino acids 244-254) was selected from the CD44H stem region.

b) Preparation of antibody-producing cells Recombinant human CD44H stem region protein 51.8 μg prepared by the method described in a) above was intraperitoneally administered to two 6-week-old BALB / c female mice together with complete Freund's adjuvant for initial immunization. After 21 days, each mouse initially immunized with 59.4 μg of recombinant human CD44H stem region protein dissolved in 0.9% sodium chloride aqueous solution was intraperitoneally administered and boosted. Further, after 34 days, 40 μg of recombinant human CD44H stem region protein was intravenously and intraperitoneally administered in the same manner as in the booster immunization to obtain the final immunization. Three days later, the spleen was removed and used as a spleen cell suspension. CD44H stem region fragment (RS1) and P1 oligopeptide were also immunized by the same method.

c) Cell fusion (1) The following materials and methods were used.
RPMI-1640 medium: RPMI-1640 (Flow Lab.) With sodium bicarbonate (24 mM), sodium pyruvate (1 mM), penicillin G potassium (50 U / ml), amikacin sulfate (100 μg / ml) and dry ice The pH was adjusted to 7.2, and sterilization filtration was performed with a 0.2 μm Toyo membrane filter.
NS-1 medium: FCS (MABioproducts) sterilized and filtered into the above RPMI-1640 medium was added to a concentration of 15% (v / v).
PEG4000 solution: Polyethylene glycol 4000 (PEG 4000, Merk & Co.) was added to RPMI-1640 medium to a concentration of 50% (w / w) to prepare a serum-free solution.
Fusion with 8-azaguanine-resistant myeloma cells SP2 (SP-2 / 0-Ag14) was performed according to Oi and described in Selected Method in Cellular Immunology, eds.BB Mishell and SM Shiigi, WH Freeman and Company, 351-372, 1980. The method of Herzenberg et al. Was slightly modified.

(2) Nucleated spleen cells (live cell rate 100%) and myeloma cells (live cell rate 100%) prepared in b) were fused at a ratio of 5: 1. Spleen cells and myeloma cells were separately washed with the above RPMI-1640 medium, then suspended in the same medium and mixed at the above ratio for fusion. Using a polypropylene centrifuge tube (Iwaki Glass) with a capacity of 250 ml, the mixture was centrifuged at 400 × g for 10 minutes in 25 ml of RPMI-1640 medium, and the supernatant was completely sucked out. To the precipitated cells, 4.3 ml of a 37 ° C. warmed PEG 4000 solution was added dropwise over 1 minute, and the mixture was further stirred for 1 minute to resuspend and disperse the cells. Next, 4.3 ml of 37 ° C. warmed RPMI-1640 medium was added dropwise over 1 minute. After repeating this operation once more, 29.2 ml of the same medium was added dropwise with constant stirring for 2-3 minutes to disperse the cells. This was centrifuged at 400 × g for 7 minutes, and the supernatant was completely removed by aspiration. Next, 37 ° C. warming and 42.7 ml of NS-1 medium were rapidly added to the precipitated cells, and a large lump of cells was dispersed by carefully pipetting with a 10 ml pipette. Furthermore, 85.3 ml of the same medium was added for dilution, and 6.0 × 10 ⁵ cells / 0.1 ml of cells were added to a 96-well polystyrene microwell (Iwaki Glass). The above microwells to which cells were added were cultured in 7% carbon dioxide gas / 93% air at a temperature of 37 ° C. and a humidity of 100%.

d) Selective growth of hybridomas on selective medium (1) Medium used is as follows.
HAT medium: Hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine (16 μM) were further added to the NS-1 medium described in c) above.
HT medium: The same composition as the HAT medium except that aminopterin was removed.
(2) On the next day (first day) after the start of culture in c), 2 drops (about 0.1 ml) of HAT medium were added to the cells with a Pasteur pipette. On days 2, 3, 5, 8, and 11, half of the medium (0.1 ml) was replaced with fresh HAT medium. Positive wells were checked by ELISA described in the next section e) for all the hybridoma-grown wells. Next, as a feeder, 1 ml of HT medium containing 10 ⁷ mouse thymocytes was added to a polystyrene 24-well cell well (Iwaki Glass), and the entire contents of each positive hybridoma detected above were transferred. This was subjected to culturing at 37 ° C. for about 1 week in the presence of 7% carbon dioxide gas as in the above c). Meanwhile, 0.5 ml of the supernatant in each well was exchanged with 0.5 ml of fresh HT medium once or twice. When hybridomas were sufficiently grown, positivity was reconfirmed by ELISA, and each was cloned by the limiting dilution method described in f) below. The residual liquid after use for cloning was transferred to a polystyrene 25 cm ² tissue culture flask (Iwaki Glass) to prepare a sample for cryopreservation.

e) Search for anti-CD44 antibody-producing hybridomas by ELISA
The method of Rennard et al. Described in Anal. Biochem., 104, 205, 1980 was slightly modified. This method is suitable for detection of hybridoma antibodies. A 96-well microtiter plate (Flow Lab.) Was coated with 100 ng of recombinant human CD44H stem region protein and then the uncoated portion was blocked with 1% BSA. A part of the supernatant of the hybridoma growth well obtained in d) was added to this and incubated at room temperature for about 1 hour. Horseradish peroxidase (HRP) -labeled goat anti-mouse immunoglobulin (Cappel Lab.) Was added as a secondary antibody and further incubated at room temperature for about 1 hour. Next, the degree of brown color produced by adding hydrogen peroxide and o-phenylenediamine as substrates was determined by measuring the absorbance at 492 nm using a microplate reader (MRP-A4, Tosoh).

f) Cloning After the operation of d), there is a possibility that two or more kinds of hybridomas are growing in each well. Therefore, cloning is performed by a limiting dilution method to obtain a monoclonal antibody-producing hybridoma. NS-1 medium Cloning medium containing 10 ⁷ mouse thymocytes as a feeder per ml was prepared, and 96, 1 and 0.5 hybridomas were added to 36 wells, 36 wells and 24 wells. . On days 5 and 12, 0.1 ml of NS-1 medium was added to all wells. The ELISA method was performed on a group in which sufficient hybridoma growth was observed 14 to 15 days after the start of cloning and the colony formation negative well was 50% or more. If all the tested wells are not positive, the number of colonies in the antibody-positive well is confirmed, and 4 to 6 wells in which one colony is confirmed are selected and recloned. Finally, a monoclonal antibody-producing hybridoma against recombinant human CD44H stem region protein was obtained.

g) In vitro and in vivo growth of monoclonal antibodies Monoclonal antibodies are grown by conventional methods. That is, each of the obtained hybridomas was cultured (in vitro growth) in an appropriate culture solution such as NS-1 medium, and a monoclonal antibody having a concentration of 10 to 100 μg / ml could be obtained from the culture supernatant. On the other hand, in order to obtain a large amount of antibody, 0.5 ml of tumor formation promoter pristane (Aldrich Chem. Co.) per mouse was intraperitoneally administered to mice (BALB / c) syngeneic with spleen cells and myeloma cells. did. After 1 to 3 weeks, 1 × 10 ⁷ hybridomas were also administered intraperitoneally, and after 1 to 2 weeks, ascites containing 1 to 4 mg / ml monoclonal antibody produced in vivo could be obtained. .

h) Heavy chain and light chain of monoclonal antibody According to the ELISA method described above, the culture supernatant of each monoclone obtained in f) above was added to a microtitration plate coated with recombinant human CD44H stem region protein. Next, after washing with PBS, isotype-specific rabbit anti-mouse Ig antibody (ZymedLab.) Was added. After washing with PBS, add HRP-labeled goat anti-rabbit IgG (H + L) antibody, and use hydrogen peroxide and 2,2'-azino-di (3-ethylbenzothiazoline sulfate) as a substrate, respectively for heavy and light chains. Was judged. For example, clone No. 285-2F12 was γ1 / κ, 284-43F1 was γ1 / κ, 268-1F5 was γ1 / κ, and 294-6F2 was γ2b / κ.

i) Purification of monoclonal antibody Each ascites obtained in g) above was fractionated with 40% saturated ammonium sulfate, and equilibrated with 1.5M glycine-NaOH buffer (pH 8.9) containing 0.5M sodium chloride for IgG1 class antibodies. The gel was adsorbed on a purified Affigel protein A (Bio-Rad Lab.) Column (φ2.5 × 5 cm), washed with the above buffer solution, and eluted with 0.1 M citrate buffer solution (pH 3.0). When fractionated into 4 ml of eluate / fraction, for example, clone No. 268-1F5 was eluted in fractions 4-5 and clone No. 294-6F2 was eluted in fractions 2-5 and purified.

[Recognition site search]
a) Immunostaining The recombinant human CD44H stem region protein prepared by the method of Kajita et al. in Example 1, a) was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) containing sodium dodecyl sulfate, followed by cell engineering. , 182, 1061-1068, 1983, Western blotting was performed according to the method of Tabe. After reacting with each monoclonal antibody, immunostaining was performed by indirect method using HRP-labeled goat anti-mouse immunoglobulin (Cappel Lab.). . An anti-His antibody was used to confirm that it was recombinant CD44H. As a result, 4 clones (clone Nos. 285-2F12, 284-43F1, 268-1F5 and 294-6F2) showed a specific reaction to the antigen (FIG. 2). Other anti-CD44 antibodies used in FIG. 2 are a rabbit polyclonal antibody (C-6) against the P1 peptide (Asp ²⁴³ -Ala ²⁵⁴ ) and a commercially available antibody 2C5.

b) Searching the recognition site of each monoclonal antibody in the CD44 molecule In order to examine the recognition site of each monoclonal antibody in the CD44 molecule, seven types of partially deleted CD44H stem region proteins (Fig. 3) were prepared, and each monoclonal antibody against each molecule The reactivity of was investigated. ^{RS2: Thr 130 -Arg 186, RS3} : Thr 130 -Gly 192, RS4: Thr 130 -Ser 209, RS5: Thr 130 -Pro 230, RS6: Thr 130 -Gln 250 and RS7: Thr ¹³⁰ -Glu ²⁶⁸ (both In SEQ ID NO: 1) (FIG. 4).
As a result, clone No. 285-2F12 is a Thr ^130- Arg ¹⁶² amino acid region, clone Nos. 284-43F1 and 268-1F5 are Asp ^206- Pro ²³⁰ amino acid regions, and clone No. 294-6F2 is an Asp ^243- Gln. A region of ²⁵⁰ amino acids was recognized. Furthermore, in order to narrow down the recognition sites of clone Nos. 284-43F1 and 268-1F5, two types of peptides, Pro ^205- Ile ²¹⁹ amino acid sequence and Pro ^220- Pro ²³⁰ amino acid sequence, were synthesized and the reactivity with each monoclonal antibody was determined. Examined. As a result, clone No. 284-43F1 recognized the Pro ^205- Ile ²¹⁹ amino acid region, and clone No. 268-1F5 recognized the Pro ^220- Pro ²³⁰ amino acid region. The recognition site of each clone is shown in FIG.

Antibody specificity
a) Reactivity with human cell line-derived CD44 In order to examine the reactivity of each monoclonal antibody against human cell-derived CD44, the expression of CD44 in 10 human tumor cell lines was examined (FIG. 6). The RT-PCR method was used as the method. Total RNA samples (3 μg) were reverse transcribed using 0.3 μg of random primers. The cDNA product solution was then amplified for 30 cycles with Taq DNA polymerase (20 cycles using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control sample). The primers are as follows.
CD44;
primer-1 (5'-AGACATCTACCCCAGCAAC-3 ') (SEQ ID NO: 2)
primer-2 (5'-CGTTGAGTCCACTTGGCTTTC-3 ') (SEQ ID NO: 3)
GAPDH;
primer-1 (5'-AAGGCTGAGAACGGGAAGCTTGTCATCAAT-3 ') (SEQ ID NO: 4)
primer-2 (5'-TTCCCGTCTAGCTCAGGGATGACCTTGCCC-3 ') (SEQ ID NO: 5)
Human pancreatic cancer (MIAPaCa-2 and PANC1), fibrosarcoma (HT1080), breast cancer (ZR-75-1, MCF-7 and MDA-MB231), bladder cancer (T24), prostate cancer (LNCAP), astrocyte type (U251) and cervical adenocarcinoma (HeLa) were obtained from the American Type Culture Collection (Rockville, MD). MIAPaCa-2, ZR-75-1 and U251 are RPMI1640 medium (Sigma Aldrich, Louis, MO) containing 10% fetal bovine serum (FBS) and kanamycin; other cell lines are 10% FBS and kanamycin or penicillin / streptomycin The cells were cultured in Dulbecco's modified Eagle's medium (Sigma Aldrich).
As a result, the expression of CD44 was confirmed in 7 types of cell lines, but the expression was very weak in the remaining 3 types. When the reactivity of each monoclonal antibody to these seven types of CD44 molecules was examined, a major band of 85-95 kDa was detected in all antibodies in pancreatic cancer MIAPaCa-2 and astrocytoma U251. Clone NO. 285-2F12, 2C5 and 284-43F1 responded to CD44 of all cell lines that expressed mRNA. The molecular weight was almost the same as MIAPaCa-2 and U251, although slight differences were seen in the cell lines. Polyclonal antibody C6 showed a similar recognition pattern, but a weak response to CD44 of breast cancer MDA-MB231 cells.
Clone Nos. 268-1F5 and 294-6F2 have other antibodies against CD44 of five cell lines (pancreatic cancer PANC1, fibrosarcoma HT1080, cervical adenocarcinoma HeLa, bladder cancer T24 and breast cancer MDA-MB231). 285-2F12, 2C5 and 284-43F1) did not react even though they reacted. Note that the 75 kDa molecule detected in clone No. 268-1F5 in all cell lines has nothing to do with CD44. This is because the 75 kDa molecule is also detected in cell lines that do not express CD44 (LNCAP, MCF-7 and ZR-75-1).
Thus, the CD44 molecule expressed in the five cell lines is probably modified differently than the other two CD44-producing cell line molecules and appears to have different reactivity to the antibody. .

b) Preparation and induction of recombinant adenovirus
CD44H cDNA (CD44MF) with c-Myc epitope at the N-terminus and FLAG tag at the C-terminus was prepared by a PCR-based method and incorporated into the vector plasmid pcDNA3.1 (+) Zeo (Invitrogen, Carlsbad, CA) . The cDNA insert was incorporated into the adenovirus Type5 gene using the Adeno-X Expression System (Clontech Laboratories, Inc., Palo Alto, Calif.). Recombinant adenovirus was amplified in human undifferentiated kidney 293 cells and purified by cesium chloride concentration gradient. Virus titer (infectious units / mL) was determined by Adeno-X Rapid Titer Kit (Clontech Laboratories, Inc.).
To confirm the effect of glycosylation on antibody reactivity, ZR-75-1 cells (3 × 10 ⁶ ) that do not express endogenous CD44H were seeded in a 100 mm dish and RPMI1640 medium containing 10% FBS and kanamycin. Cultured for 24 hours. Cells were infected with recombinant adenovirus in DMEM medium containing 5% FBS for 1 hour at 37 ° C. The medium was then replaced with serum-free RPMI 1640 medium and cultured for 48 hours. Cells were lysed with RIPA buffer (50 mmol Tris-HCl pH 7.5, 1% TritonX-100, 1% deoxycolic acid, 0.1% SDS, 150 mmol NaCl) containing protease inhibitors (Roche Diagnostics, GmbH). Using anti-FLAG M2 antibody-conjugated agarose beads (Sigma Aldrich), CD44 was purified by immunoprecipitation with FLAG Tag from the cell disruption extract, and immunostained by Western blotting using the purified product as an antigen. (FIG. 7). CD44MF expressed in the cells was confirmed by Western blot using anti-c-Myc antibody and anti-FLAG antibody. Its molecular weight was the same as that of the endogenous CD44 molecule expressed in the cell line of FIG. Clone Nos. 285-2F12, 284-43F1 and 2C5 detected CD44MF, but 268-1F5 and 294-6F2 did not react. Polyclonal antibody C6 reacted very weakly with CD44MF. Thus, CD44MF expressed in cells is thought to have undergone the same modification as the endogenous CD44 molecule expressed in the five cell lines used in FIG.

c) Effect of sugar chain modification on CD44 molecule The effect of post-translational modification of CD44 on the reactivity of clones Nos. 268-1F5 and 294-6F2, especially the sugar chain modification, was investigated. CD44 purified by the method described in b) above was dissolved in 50 μL of 20 mmol phosphate buffer (pH 7.0), and 2 mU neuraminidase, 0.2 mU O-glycosidase and / or 0.3 U N-glycosidase F (Roche Diagnostics, GmbH) , Mannheim, Germany) at 37 ° C. for 16 hours. Then, the change in reactivity of each antibody was confirmed by Western blotting (FIG. 8). Neuraminidase is an exoglycosidase for sialic acid located at the end of N-linked and O-linked sugar chains. O-glycosidase digests the disaccharide Galβ (1-3) GalNAc from O-linked sugar chains modified to serine or threonine residues. When sialic acid is added to the terminal, the function of O-glycosidase is inhibited. Therefore, a combination of neuraminidase and O-glycosidase is necessary to effectively hydrolyze the O-linked sugar chain. N-glycosidase F is an endoglycosidase that cleaves all types of N-linked glycans modified with asparagine. A slight change was seen in the molecular weight of neuraminidase-treated CD44MF compared to the untreated CD44MF molecule detected in clone No. 285-2F12, and further changes were seen by simultaneous treatment with O-glycosidase. However, O-glycosidase alone did not change the molecular weight of CD44MF. Treatment with N-glycosidase F reduced the molecular weight of CD44MF to 70 kDa. However, further treatment with neuraminidase did not show any further changes. The molecular weight of CD44MF treated with all enzymes simultaneously was approximately 69 kDa.
First, the reactivity of clone No. 268-1F5 to the glycosidase-treated sample was confirmed (FIG. 8). Treatment with neuraminidase or O-glycosidase alone showed no change in reactivity, but both responded to CD44MF treated simultaneously. This indicates that the O-linked sugar chain with sialic acid, probably modification near the recognition site of clone No. 268-1F5, inhibits antibody binding. Similar results were obtained when endogenous CD44 of HT1080 that did not react with clone No. 268-1F5 was treated with glycosidase. The reactivity of polyclonal antibody C-6 was enhanced by treatment with N-glycosidase F, but the change with the combination of neuraminidase and O-glycosidase was only slight. There is an N-linked sugar chain-modifiable site (NTT ²⁵⁷ ) immediately downstream of the C-6 epitope, and modification of this site may affect the reactivity of the antibody.
In contrast, the reactivity of clone No. 294-6F2 was not restored by any enzyme treatment on CD44MF. Treatment of CD44MF with three glycosidases does not completely remove the glycosylation, as can be seen from the molecular weight, so the remaining modifications may cover the reactive site of the antibody. In particular, O-glycosidase cannot cleave parts such as GlucNac and GalNac of O-linked sugar chains.

d) Examination of amino acid sequence affecting immune response Next, the site affecting the immunoreactivity of clone No. 294-6F2 was examined. A serine-threonine rich sequence (SHTT ²³⁷ ) containing an O-linked sugar chain-modifiable site can be seen near the recognition site of clone No. 294-6F2. In order to evaluate the effect of modification at this site, using the GeneEditor site-directed mutagenesis system (Promega), the 234th serine, 236th and 237th threonine residues which are sugar chain-modifiable sites are alanine as shown in FIG. Substituted with a residue. Next, mutant expression plasmids were introduced into ZR-75-1 cells using FuGENE6 ^™ (Roche Diagnostics, GmbH), the cells were cultured, the mutants were expressed, and immunostaining was performed (FIG. 9). As a result, all mutants were expressed in ZR-75-1 cells and could be detected by Western blot with clone No. 285-2F12. Mutants in which wild type CD44MF and 236th threonine were substituted with alanine (SHAT, Mut.SAT) did not change the reactivity. However, mutants with substitution of 234th serine / 236-237th threonine (AHAA, Mut.AAA), mutants with substitution of 236-237th threonine (SHAA, Mut.SAA), or mutants with substitution of 237th threonine (SHTA, Mut.STA) was recognized by clone No. 294-6F2. This suggests that the 234th serine and 237th threonine residues in the vicinity of the antibody recognition site are important for reactivity and are probably modified by O-linked sugar chains. The effects of these mutants were specific to clone No. 294-6F2, and did not affect the reactivity of clone No. 268-1F5.

[Tissue staining]
a) Cell staining Human breast cancer cells ZR-75-1 cells (American Type Culture Collection) were infected with CD44 gene-introduced adenovirus, cultured overnight on fibronectin-coated cover glass, and cells were allowed to adhere to express CD44. . After removing the culture supernatant, the cells were gently washed with PBS and immersed in 3% paraformaldehyde-containing PBS for 10 minutes to fix the cells at room temperature. After washing with PBS, 1 μg / mL of each antibody solution was added and allowed to stand at room temperature for 2 hours. Thereafter, the antibody solution was removed, lightly washed with PBS, PBS containing 1 μg / mL Alexa488-labeled goat anti-mouse IgG (Molecular Probes) was added, and the mixture was allowed to stand at room temperature for 1 hour. After removing the antibody solution and washing with PBS, the cells were observed with a Bio Rad MRC-1024 conforcal laser microscope (Bio Rad). Whole cells were visualized by staining F-actin with Alexa568 labeled phalloidin (Molecular Probes, OR). As a result, clones Nos. 284-43F1, 268-1F5 and 294-6F2 showed a significant reaction in CD44-expressing ZR-75-1 cells compared to ZR-75-1 cells. However, in clone No. 285-2F12, no reactivity could be confirmed in any cell. 268-1F5 reacted with a 75 kDa molecule that was unidentified by Western blot. However, immunostaining examined CD44 negative cells (ZR-75-1), but no signal was detected.
A human tumor cell line (MIAPaCa-2) expressing CD44 was cultured on a cover glass for 20 hours. Cells were fixed with 3% paraformaldehyde, and anti-CD44 antibody was reacted with CD44. As a result, clones Nos. 284-43F1, 268-1F5 and 294-6F2 reacted with endogenous CD44 expressed by MIAPaCa-2 and stained cells.

b) Tissue staining Human laryngeal cancer and breast cancer tissues were fixed with a periodate-lysine-paraformaldehyde solution and embedded in paraffin. Tissue sections were treated with each antibody solution (10 μg / mL), further treated with biotin-labeled equine anti-mouse IgG (Vector Laboratories) and avidin-biotin-HRP complex solution (DAKO), and 3,3'- The color was developed with diaminobenzidine tetrahydrochloride (Sigma Aldrich) and observed with a microscope. As a result, tumor cells and some stromal cells were stained with clone No. 284-43F1. Stromal cells look like blood cells infiltrating the gaps in the tumor tissue. This is because it is known that its cell form is different from that of fibroblasts and that CD44H is expressed in lymphocytes and histocytes (macrophages). A similar staining pattern was obtained with antibody 2C5. In contrast, clones Nos. 268-1F5 and 294-6F2 showed cell type-specific differences in staining. Clone No. 268-1F5 stained only tumor cells in both breast and laryngeal cancer tissues. On the other hand, clone No. 294-6F2 stained only tumor cells in breast cancer tissue and only stromal cells in laryngeal cancer tissue. Since these signals are absorbed by the oligopeptide containing the epitope sequence, they are specific to the epitope sequence. Thus, the reactivity of clones Nos. 268-1F5 and 294-6F2 is clearly different between tumor cells and stromal cells in the tumor tissue. Since the reactivity of these antibodies is affected by sugar chain modification, it is considered that the sugar chain modification of CD44H in the vicinity of the antibody recognition site controls the cell type-specific manner in tumors.

[Immunological assay]
a) Preparation of monoclonal antibody-bound solid phase carrier
According to the method of Ishikawa et al. Described in J. Immunoassay, 4, 209-327, 1983, clone No. 285-2F12 was dissolved in 0.1 M phosphate buffer (pH 7.5) and adjusted to a concentration of 100 μg / ml. did. The monoclonal antibody solution was added to a 96-well microplate at 100 μl per well and allowed to stand at 4 ° C. for 24 hours. Next, the monoclonal antibody solution was removed, washed 3 times with physiological saline, and then immersed in 30 mM phosphate buffer (pH 7.0, buffer A) containing 1% BSA, 0.1 M sodium chloride and 10 mM EDTA. Stored at ° C.

b) Preparation of enzyme-labeled antibody (IgG-HRP complex)
An anti-CD44H IgG-HRP complex was prepared according to the method of Ishikawa et al. Described in J. Immunoassay, 4, 209-327, 1983. A monoclonal antibody (IgG: clone No. 268-1F5) is dialyzed against 0.1M phosphate buffer (pH 6.5), and 100-fold mol S-acetylmercaptosuccinic anhydride is added to IgG contained in the solution. Was added as a dimethylformamide solution and incubated at 30 ° C. for 30 minutes. Next, 0.1M Tris-HCl buffer (pH 8.0) was 1/5 of the total volume, 0.1M EDTA solution (pH 6.0) was 1/50 of the total volume, and 1M hydroxylamine solution (pH 7.0). ) Was added to 1/5 of the total volume, allowed to stand at 30 ° C for 5 minutes, gel filtered with Sephadex G-25 equilibrated with 5 mM EDTA-containing O.1M phosphate buffer (pH 6.0), and SH Group-labeled anti-CD44 IgG was obtained. Preparation of maleimide-labeled HRP Dissolve HRP in a 0.1 M phosphate buffer (pH 7.0) to a concentration of 10 mg / ml, add 25-fold molar amount of EMCS to the amount of HRP as a dimethylformamide solution, The reaction was carried out at 30 ° C. for 30 minutes. This reaction solution was subjected to gel filtration with a Sephadex G-25 column equilibrated with 0.1 M phosphate buffer (pH 6.0) to fractionate the maleimide-labeled HRP fraction. About 1 mol of maleimide-labeled HRP was added to 1 mol of SH group-labeled IgG, and the mixture was allowed to stand at 4 ° C. for 20 hours. This mixed solution was subjected to gel filtration with an Ultrogel AcA44 column (Villeneuve-la-Garenne, France) equilibrated with 0.1 M phosphate buffer (pH 7.0) to fractionate an anti-CD44H IgG-HRP complex fraction. BSA and chlorhexidine were added to 0.1% and 0.002%, respectively, and stored at 4 ° C.
Similarly, an anti-CD44H IgG-HRP complex was prepared using a monoclonal antibody (IgG: clone No. 284-43F1).

c) Two-step sandwich EIA method
Recombinant CD44H (rCD44H) purified by anti-FLAG antibody-binding column, etc., expressing CD44H cDNA with FLAG tag added to CHO cells, or the culture supernatant of the cell line examined in Example 3a) was added to a 96-well vinyl plate (Falcon) Diluted 4 times with 10 mM TCEP (PIRCE), 1% BSA, 0.1 M sodium chloride and 10 mM EDTA in 30 mM phosphate buffer, pH 7.0, and left at 25 ° C. for 30 minutes. 100 μL was added to the antibody-binding plate prepared in a) and reacted at 25 ° C. for 1 hour. Next, after washing with physiological saline three times, the enzyme-labeled antibody prepared in b) above is 1 μg / mL for immune reaction buffer (1% BSA, 0.1 M sodium chloride and 10 mM EDTA-containing 30 mM phosphoric acid). Dilute with buffer solution (pH 7.0), add 100 μL each, and mix. After reacting for 1 hour at room temperature, it was washed 3 times with physiological saline. Next, 100 μL of 0.1 mg / mL tetramethylbenzidine (TMB) solution was added per well, reacted at room temperature for 30 minutes, and then 100 μL of 1 mol / L sulfuric acid was added to stop the reaction. A _{450 of} this reaction mixture was measured using a microplate reader (MPR-A4, Tosoh), and the amount of CD44 in the sample was determined from a calibration curve (FIG. 10, Table 1).

In the present invention, a tool is provided to help clarify the glycosyltransferase / addition enzyme involved in the sugar chain modification of CD44. It is possible to study and elucidate how it works in biological tissues including In addition, it is possible to elucidate and study what biological activities are involved in alternative splicing (conjugation), glycosylation, and shedding (cleaving / release) occurring in the stem region of CD44.
It will be apparent that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and thus are within the scope of the claims appended hereto.

<Sequence Listing Free Text>
SEQ ID NO: 2, Oligonucleotide to act as a primer for PCR
SEQ ID NO: 3, Oligonucleotide to act as a primer for PCR
SEQ ID NO: 4, Oligonucleotide to act as a primer for PCR
SEQ ID NO: 5, Oligonucleotide to act as a primer for PCR

The schematic diagram of an antigen is shown. CD44H is shown on the top, and the polypeptide used for the immunogen is shown on the bottom (CD44H stem fragment, sCD44HS; N-terminal fragment, RS1; C-terminal peptide, P1). CD44H is composed of a hyaluronic acid binding region (HA binding), a stem sequence (Stem), a transmembrane region (TM), and an intracellular region (CP). rCD44HS (hereinafter aa130-268 in SEQ ID NO: 1, respectively) has FLAG at the N-terminus and His6 at the C-terminus. RS1 (aa130-162) has a GST-tag at the N-terminus. P1 contains 9 amino acids from positions 243-254. Antibody reactivity is shown. To examine the reactivity of the prepared antibody, E. coli cell lysate containing rCD44HS was used for Western blot. 285-2F12, 284-23F1, 268-1F5 and 294-6F2 shown in the figure are prepared monoclonal antibodies. C-6 is a polyclonal antibody against the P1 peptide. Anti-His is an antibody against His-tag, and 2C5 is an anti-CD44 antibody commercially available from R & D Systems Inc. (MN, USA). Figure 3 shows sequence deletion mutants for epitope mapping. Six fragments of a partial sequence excised from the C-terminal side of rCD44HS are shown (RS2-7). Each fragment (RS2-7) contains aa130 to 186, 192, 209, 230, 250 and 268 in SEQ ID NO: 1. The reactivity of the antibody to the stem region fragment whose sequence has been excised is shown. A sample of E. coli cell lysate containing any one of the sequence excised fragments was applied to a Western blot using antibody (5 μg / mL). The amino acid sequence recognized by the antibody is shown. The amino acid sequence of the CD44H stem region is shown, and the sequence including the recognition site is shown in a box. The detection result of CD44 expressed in the human tumor cell line by anti-CD44 antibody is shown. Human pancreatic cancer (MIAPaCa-2, PANC1), astrocytoma (U251), fibrosarcoma (HT1080), bladder cancer (T24), prostate cancer (LNCAP), cervical adenocarcinoma (HeLa) and breast cancer (ZR-75) -1, MCF-7, MDA-MB231) cells were lysed in 2-mercaptoethanol-containing SDS-PAGE loading buffer and applied to Western blot analysis using anti-CD44 antibody and anti-actin antibody (3 × 10 ⁴ cells) /lane). The gene expression of CD44H and GAPDH was confirmed by RT-PCR. The expression and detection result of CD44H in ZR-75-1 cells are shown. Human breast cancer ZR-75-1 cells were infected with a recombinant adenovirus introduced with the CD44H gene to express CD44H with a FLAG tag. Cells were lysed in RIPA buffer and CD44H was recovered by immunoprecipitation using an anti-FLAG antibody. The precipitate was applied to SDS-PAGE and analyzed by Western blot using the indicated antibody (5 μg / mL). The influence of the sugar chain on the reactivity of the antibody to CD44H is shown. CD44H was expressed by infecting ZR-75-1 cells with CD44MF adenovirus. The CD44MF 90 kDa molecule was obtained from lysate by immunoprecipitation. Samples were treated with 2mU neuraminidase, 0.2mU O-glycosidase and / or 0.3U N-glycosidase F at 37 ° C for 16 hours, using anti-CD44 antibodies (285-2F12, 268-1F5, 294-6F2 and C-6) Applied to Western blot analysis. The schematic diagram of the CD44H S / T site substitution mutant and the effect of mutation of the O-linked sugar chain-modifiable site located near the epitope of clone No. 294-6F2 are shown. Serine residue 234 and threonine residues 236 and 237 were replaced with alanine (*). The sequence in the box indicates the epitope site of the anti-CD44 antibody, clone No. 294-6F2. CD44H (Wild type), S / T site substitution mutants (Mut.AAA, SAT, STA and SAA) and control vector (Mock) are expressed in ZR-75-1, anti-CD44 antibody, clone No. 285-2F12 And 294-6F2. The measurement result of CD44 by the immunoassay is shown. Recombinant CD44H is used at a concentration of 2.5 to 160 ng / mL, clone No. 285-2F12 is used as the solid phase, clone No. 268-1F5 is used as the labeled antibody, and a two-step ELISA is performed to measure the absorbance at a wavelength of 450 nm. Then, a calibration curve was drawn in double logarithm.

Claims (24)

An anti-CD44 antibody characterized by specifically recognizing a site consisting of a continuous amino acid sequence present in an inner extracellular domain of the extracellular domain of CD44 protein (however, the amino acid represented by SEQ ID NO: 1) Except for an anti-CD44 antibody characterized by specifically recognizing a continuous amino acid sequence site of Thr ¹⁶³ -Arg ¹⁸⁶ present in the sequence). The anti-CD44 according to claim 1, wherein the inner extracellular domain is a stem region of the extracellular domain and a region contiguous to the stem region and immediately preceding the transmembrane domain (TM). antibody. The inner extracellular domain consists of amino acid residues up to Cys ¹²⁹ on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1 of CD44H protein (including the 20 amino acid residues of the signal peptide) The continuous amino acid sequence found in a region from the amino acid residue of Thr ¹³⁰ adjacent to the globular region to the 268th amino acid residue Glu counted from the N-terminal side thereof, or 2. The anti-CD44 antibody according to 2. 2. The anti-CD44 antibody according to claim 1, wherein the portion consisting of a continuous amino acid sequence present in the inner extracellular domain of the extracellular domain of CD44 protein is at least 2 to 159 amino acid residues. . 2. The anti-CD44 antibody according to claim 1, wherein the portion consisting of a continuous amino acid sequence present in the inner extracellular domain of the extracellular domain of CD44 protein is at least 4 to 50 amino acid residues. . It is characterized by specifically recognizing the continuous amino acid sequence found in the region immediately before the transmembrane domain (TM) in the stem region and the contiguous region of the CD44 protein, and is sensitive to glycosylation. Anti-CD44 antibody. The anti-CD44 antibody according to claim 6, wherein the sugar chain modification of the CD44 protein is an O-linked sugar chain modification. The anti-CD44 antibody according to claim 6 or 7, wherein the sugar chain modification of the CD44 protein is an O-linked sugar chain modification with sialic acid. The anti-CD44 antibody according to any one of claims 6 to 8, wherein binding to the CD44 protein is inhibited by modification of the sugar chain of the CD44 protein. The anti-CD44 antibody according to any one of claims 6 to 9, which is reactive with tumor cells but non-reactive with stromal cells in cancer tissue. SEQ ID NO CD44H protein: any of claims 6-10, characterized in that to recognize Pro ²²⁰ -Pro ²³⁰ of 1 amino acid sequence represented by (counting, including 20 amino acid residues of the signal peptide) The anti-CD44 antibody according to one. The contiguous amino acid sequence of Ser ²³⁴ -Thr ²³⁷ of the amino acid sequence represented by SEQ ID NO: 1 of CD44H protein (including the 20 amino acid residues of the signal peptide) inhibits binding to CD44 protein. The anti-CD44 antibody according to claim 6. 13. The anti-antibody according to claim 6 or 12, which recognizes Asp ²⁴³ -Gln ²⁵⁰ of the amino acid sequence represented by SEQ ID NO: 1 of CD44H protein (counted including 20 amino acid residues of the signal peptide). CD44 antibody. The anti-CD44 antibody according to claim 13, wherein the reactivity with the CD44 protein is changed by sugar chain modification in the vicinity of the epitope. The anti-CD44 antibody according to any one of claims 1 to 5, which is not affected by O-linked sugar chain modification of CD44 protein. CD44 protein SEQ ID NO: 1 amino acid sequence represented by the claims 1 to 5 and 15 and recognizes the Pro ²⁰⁵ -Ile ²¹⁹ in the (counting, including 20 amino acid residues of the signal peptide) The anti-CD44 antibody according to any one of the above. CD44 protein SEQ ID NO: any one of the preceding claims, characterized in that to recognize Thr ¹³⁰ -Arg ¹⁶² of 1 represented by the amino acid sequence (counting, including 20 amino acid residues of the signal peptide) The anti-CD44 antibody according to one. The anti-CD44 antibody according to any one of claims 1 to 17, wherein the antibody is a monoclonal antibody. A method for discriminating a CD44 sugar chain, comprising detecting a difference in sugar chains of CD44 expressed on a cell using the anti-CD44 antibody according to any one of claims 1 to 17. A method for discriminating a cell sugar chain in a tumor tissue, comprising detecting a difference in sugar chain modification of the cell in the tumor tissue using the anti-CD44 antibody according to any one of claims 1 to 17. 21. The method of claim 20, wherein the cell is a tumor cell. A method comprising distinguishing tumor cells and non-tumor cells in a tumor tissue using the anti-CD44 antibody according to any one of claims 1 to 17. 23. The method of claim 22, wherein the non-tumor cell is a stromal cell. Containing the anti-CD44 antibody according to any one of claims 1 to 17 as an active ingredient,
(1) Detecting the difference in the sugar chain of CD44 expressed on cells,
(2) detecting differences in cell glycosylation in tumor tissue, and
(3) An anti-CD44 antibody reagent which is used for being selected from the group consisting of: distinguishing tumor cells and non-tumor cells in tumor tissue.

Images