JP2009067678A - Antibody against connective tissue growth factor or composition containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody having an ability for specifically recognizing a connective tissue growth factor (CTGF) and for inhibiting a biological activity thereof. <P>SOLUTION: The antibody can specifically recognize CTGF, especially a high-order structure of the natural type of CTGF, and is prepared by the immunization of DNA, including immunizing a non-human animal with an expression vector containing a DNA encoding CTGF or its functional fragment in an expressible state. The antibody is usable as a composition for treating, preventing or diagnosing diseases relating to the antibody, such as fibrosis or cell proliferative diseases. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an antibody capable of specifically recognizing connective tissue growth factor (CTGF).

  The present invention also relates to a method for producing the antibody, and a pharmaceutical composition or diagnostic composition containing the antibody.

  Human CTGF is a connective tissue growth factor with 39 cysteine residues consisting of 322 amino acids (but 349 amino acids when including a signal peptide), and its amino acid and base sequence have been known since 1991 (non- Patent Documents 1-2). While this protein is known to have a wound healing action, it is known to be related to diseases and disorders such as cell proliferative diseases, fibrotic diseases, atherosclerosis, glaucoma (Patent Documents 1 to 7, Non-patent documents 2 to 4). For the treatment or diagnosis of such diseases and disorders, the use of antibodies against human CTGF has been proposed (Patent Documents 1 to 7, Non-Patent Document 4).

  In general, a method of producing an antibody by immunizing an animal with a protein or peptide as an antigen is used to produce the antibody. In vivo antibody production is achieved by the immunized animal recognizing the antigen as a foreign substance, presenting the antigen to the antigen-presenting cell, signaling it to the T cell, and activating it. Is further performed by stimulating B cells and directing them to antibody producing cells.

  However, attempts by the present inventors to produce antibodies using conventional antibody production methods that directly immunize animals with human CTGF as an antigen have often failed or have very low antibody titers. there were. This was also observed when, for example, human CTGF was immunized not only in mammals but also in chickens. The cause is not clear, but CTGF is inherently a strong protein, so it is presumed that it adhered to various surfaces and as a result, sufficient immunization was not performed.

  Human CTGF also has the unique structure inherent to this protein, that is, it has 39 cysteine residues and has at least 5 disulfide bonds, making it susceptible to recombination of intramolecular disulfide bonds. Have Therefore, when this protein is exposed to a non-natural environment (for example, a purification environment), it is presumed that such recombination occurs, thereby changing the protein's original higher-order structure, resulting in protein denaturation. The One of the future tasks is to make it possible to obtain an antibody that specifically recognizes the natural higher-order structure of human CTGF due to such special structural characteristics.

  Although there are patent documents and publications related to antibodies against human CTGF as described above, there is no method for producing such antibodies in large quantities, and antibodies that specifically recognize their natural higher-order structures. Since it is difficult to actually obtain the antibody, no practical antibody has yet appeared even though it is desired.

  In the present application, a method for producing an antibody using a DNA immunization method (or gene immunization method) is provided. The DNA immunization method is described in, for example, Patent Document 8 and Non-Patent Documents 5-8. The DNA immunization method is a so-called genetic vaccine in which a DNA encoding an antigen is inserted into a vector so that an antibody against a certain antigen is produced in vivo, and the vector is inoculated into an animal such as a human. Refers to inoculation. Such antigens include, for example, viral antigens, tumor associated antigens, protozoan antigens, microbial antigens. After inoculation, DNA is expressed, antigens are generated, and taken up into the in vivo immune system to produce antibodies.

As an application of DNA immunization, Non-Patent Document 9 describes the production of polyclonal antibodies using DNA immunization. This method includes incorporating a DNA encoding a fusion protein such as a secretory leader and an antigenic tag linked to the N-terminal side of the antigen protein into a vector so as to allow expression, and immunizing the animal with the vector. Antigenic tags act like adjuvants and generate antibodies against the tags in vivo.
Japanese National Patent Publication No. 11-507332 Japanese Patent Laid-Open No. 11-180895 JP 2004-132974 A Special table 2002-529066 gazette Special Table 2002-504332 Special Table 2005-505757 Special Table 2002-524422 Special Table 2000-582427 DM Bradham et al., J. Cell Biol. 114: 1285-1294 (1991) A. Igarashi et al., Mol. Biol. Cell 4: 637-645 (1993) M. Minato et al., J. Biochem. 135: 347-354 (2004) T. Mori et al., J. Cellular Physiology 181: 153-159 (1999) JS Boyle et al., Proc. Natl. Acad. Sci. USA 94: 14626-14631 (1997) S. Gurunathan et al., Annu. Rev. Immunol. 18: 927-974 (2000) DC Tang et al., Nature 356: 152-154 (1992) H. Tighe et al., Immunol. Today 19: 89-97 (1998) RS Chambers and SA Johnston, Nature Biotechnology 21 (9): 1088-1092 (2003)

  Human CTGF belongs to the CCN family and is known to have various biological functions as one of growth factors formed by four modules. In particular, CTGF is attracting attention as a pre-curing molecule in fibrosis such as dermal fibrosis, and the development of specific antibodies is a study to investigate the relationship between the expression of this molecule and the progression of disease state, and overproduction of CTGF including fibrosis It is considered to be an important key for the development of molecular targeted therapy for diseases related to the disease. However, it is difficult to produce and purify the molecule as a recombinant protein in large quantities for the purpose of securing an antigenic protein that retains its activity, and it is extremely difficult to produce antibodies against CTGF molecules by conventional antibody production methods. It was difficult. In fact, commercially available CTGF antibodies exist but are not suitable for use due to their low specificity.

  Under such circumstances, an object of the present invention is to provide an antibody capable of specifically recognizing natural CTGF.

  Another object of the present invention is to provide a pharmaceutical composition or a diagnostic composition comprising the antibody.

  Still another object of the present invention is to provide a method for producing the above antibody.

  The present inventors initially produced recombinant human CTGF that was expressed using insect cells infected with recombinant baculovirus, and that expressed DNA encoding human CTGF, and purified using an affinity column. Mice and chickens were immunized but the expected antibody was not produced. In order to obtain an antibody that recognizes the protein having the natural higher-order structure, the antibody production in mice was next attempted by DNA immunization. As a result of antiserum analysis of the mice by flow cytometry, the antibody titer was It was confirmed to be low.

  Furthermore, the present inventors inserted DNA encoding an immunopotentiating signal peptide into an expression vector in the DNA immunization method, and examined the increase in antibody titer due to immunization. However, no significant increase in CTGF antibody titer was observed. The cause was expected to be insufficient for the induction of the immune system because the CTGF antigen concentration as an expression product was very low. CTGF is a highly sticky protein, and these characteristics may have influenced it. Therefore, considering that CTGF stays on the cell surface at a high concentration for a certain period of time, a plasmid with a DNA encoding a localization signal added just before the immune enhancement signal was prepared and immunized in the same way. In addition to succeeding in producing a practical CTGF antibody, it has become possible to provide an antibody that specifically recognizes the natural human CTGF structure with a higher affinity than ever before. Human CTGF is a protein with unique properties both in terms of structure and high adhesiveness. Until now, antibodies against CTGF have been commercially available, but they are antibodies that are so low in specificity that they cannot be used. In addition, because of its unique properties, CTGF has the property that antibodies are extremely difficult to obtain, so that the methods disclosed in conventional patent documents and publications do not yield antibodies, or Only a very low antibody titer and an impractical antibody were obtained. In contrast, the method of the present invention makes it possible to produce human CTGF antibodies that are reproducible and specific. Such a method has not been conceived by those skilled in the art until now, and it has become possible to produce antibodies at a practical level and to supply antibodies that specifically recognize natural human CTGF. Is groundbreaking.

Therefore, in summary, the present invention has the following features.
In the first aspect, the present invention provides an antibody produced by DNA immunization or a fragment thereof capable of specifically recognizing connective tissue growth factor (CTGF).

In one embodiment thereof, the CTGF is human CTGF.
In another embodiment thereof, the antibody of the present invention is a human antibody or a humanized antibody.

  In this specification, a human antibody refers to a fully human antibody and is the same antibody as the antibody produced by a human antibody gene. The humanized antibody is a chimeric antibody derived from a part of a human antibody and a part of an antibody of another animal. For example, a part or all of the CDR of the hypervariable region of a human antibody is a non-human animal. An antibody that is substituted for its counterpart (eg mouse).

  In another embodiment thereof, the DNA immunization includes immunizing a non-human animal with an expression vector that can express the DNA encoding the CTGF or a functional fragment thereof.

The vector can further include DNA encoding a secretory signal peptide.
The vector can further include DNA encoding a peptide that includes a localization signal and an immune enhancement signal.

  Preferably, the vector contains a DNA encoding each of a secretion signal peptide, a localization signal peptide, an immune enhancement signal peptide, and a CTGF protein in this order from the 5 ′ end side.

  As used herein, a secretory signal peptide is a peptide that functions to transport CTGF expressed and translated in cells to the cell membrane, and is a peptide that is cleaved by processing with a signal peptidase. At this time, CTGF is secreted extracellularly. A preferred peptide is the CTGF signal peptide.

  As used herein, a localized signal peptide is any peptide having a function of transferring CTGF expressed and translated in cells to the cell membrane and anchoring secreted CTGF on the cell membrane.

  As used herein, an immunoenhancing signal peptide is any peptide that enhances the immunogenic or antigenic activity of CTGF.

  In the present invention, an example of the functional fragment is a CTGF module. Here, the module refers to a component having a function, function or action in CTGF protein. Examples of such modules are insulin-like growth factor binding elements, von Willebrand C type elements, thrombospondin type 1 repeat elements, C-terminal cysteine knot elements, heparin binding elements, and the like. Specifically, it includes module 1, module 2, module 3 or module 4 of human CTGF as shown in FIG.

  As used herein, the term DNA includes both cDNA and genomic DNA. cDNA can be synthesized by cDNA cloning from mRNA in animal tissues or cells.

In another embodiment thereof, the DNA encoding the full-length human CTGF or a functional fragment thereof comprises a base sequence represented by SEQ ID NO: 2, 4, 6, 8 or 10.
In another embodiment thereof, the animal is a mammal.
In another embodiment thereof, the mammal is a mouse.

  Preferably, the mouse is a human antibody-producing transgenic mouse. This mouse is a transgenic mouse in which a human antibody gene is introduced in place of the mouse antibody gene on the mouse chromosome by a gene recombination technique using a human artificial chromosome, whereby a fully human antibody can be produced.

  In another embodiment thereof, the antibody of the present invention can specifically recognize the natural conformation of the CTGF molecule.

  In this specification, the natural higher-order structure of CTGF is the native higher-order structure taken in vivo by CTGF formed by disulfide bonds at natural positions formed between rich cysteine residues in the amino acid sequence. (Also referred to as conformation).

  Further, in the present specification, the specific recognition of CTGF's natural conformation means that the antibody of the present invention represents a continuous or discontinuous surface antigen epitope of CTGF having a natural conformation. It means better recognition compared to the surface antigen epitope of denatured CTGF that has undergone structural modification by intramolecular disulfide bond recombination during the purification or gene recombination of CTGF. Here, specific means that CTGF having a natural higher-order structure is recognized but the above-mentioned modified CTGF is not recognized, or CTGF having a natural higher-order structure is relatively recognized than the above-mentioned modified CTGF. Means easy. Such a relatively high recognition degree (or immunological reactivity), in other words, a binding ability that is 10 times or more, preferably 100 times or more, more preferably 1,000 times or more is specifically mentioned in the present specification. Recognize.

  In the present specification, DNA immunity is expressed and secreted intracellularly by injecting an expression vector incorporating a CTGF-encoding DNA or a functional fragment thereof into a non-human animal, preferably a mammal, subcutaneously. Furthermore, due to the heterogeneity of the protein, it means a series of procedures including the production of antibodies against the protein in animals.

  The finding that the antibody of the present invention can be obtained by using the above-described DNA immunization method rather than the conventional antibody production method in which a proteinaceous or peptide antigen is directly injected subcutaneously into an animal is the feature of the present invention. It is also one, which was not anticipated by those skilled in the art before filing this application.

  In the present specification, “expressible” means that the DNA encoding CTGF or a functional fragment thereof is expressed under the control of a regulatory sequence, and a corresponding protein or peptide is generated in vivo.

  In another embodiment thereof, the antibody of the present invention can specifically recognize any one of the module 1, module 2, module 3 or module 4 of the human CTGF.

In another embodiment thereof, the antibody of the present invention has a binding constant (KA) for CTGF of 3.0 × 10 ⁷ M ⁻¹ or more and / or a dissociation constant (KD) of 5.6 × 10 ⁻⁹ M or less.

  The binding constants of antibodies produced by conventional immunization methods (polyclonal or monoclonal) to native human CTGF have not been specifically reported so far, but such antibodies are difficult to cross-react with human CTGF and have low affinity. It is known (eg J. Biochem, 2004, 135: 347-354). All of the antibodies of the invention are characterized by having a higher binding constant for human CTGF or a lower dissociation constant than antibodies produced by conventional immunization methods.

  The present invention also provides, in a second aspect, a pharmaceutical composition comprising an antibody of the present invention or a fragment thereof as described above.

In one embodiment thereof, the antibody of the present invention is a monoclonal antibody.
In another embodiment thereof, the antibody of the present invention is a human antibody or a humanized antibody.
In another embodiment thereof, the antibody of the invention is conjugated to a therapeutic agent.

  Such a conjugate of an antibody and a therapeutic agent is a medicine intended for so-called molecular targeting. The antibody and therapeutic agent of the present invention are transported to a disease target site by the antibody and can act for the treatment or prevention of diseases related to CTGF.

  Examples of such diseases include fibrosis or sclerosis associated with fibrosis, or cell proliferative diseases. Specifically, the sclerosis associated with fibrosis or fibrosis includes, for example, renal fibrosis, pulmonary fibrosis, liver fibrosis, or scleroderma (or dermal fibrosis). The cell proliferative disease is, for example, cancer.

  In the third aspect, the present invention further provides a diagnostic composition comprising the antibody of the present invention or a fragment thereof as described above.

In one embodiment thereof, the antibody of the present invention is a monoclonal antibody.
In another embodiment thereof, the composition of the present invention is used for diagnosis of fibrosis or sclerosis associated with fibrosis, or cell proliferative disease.

  The fibrosis associated with fibrosis or fibrosis is, for example, renal fibrosis, pulmonary fibrosis, liver fibrosis or scleroderma (or dermal fibrosis). The cell proliferative disease is cancer.

  In another embodiment thereof, the antibody of the present invention is mouse-mouse hybridoma CTGF-m11 (FERM ABP-10454), mouse-mouse hybridoma CTGF-m12 (FERM ABP-10455) or mouse-mouse hybridoma CTGF-m21 (FERM ABP-10456) is a monoclonal antibody derived from a hybridoma.

  In the fourth aspect, the present invention further provides a monoclonal antibody derived from a Mouse-Mouse hybridoma CTGF-m11 (FERM ABP-10454) hybridoma.

  In the fifth aspect, the present invention further provides a monoclonal antibody derived from a Mouse-Mouse hybridoma CTGF-m12 (FERM ABP-10455) hybridoma.

  In the sixth aspect, the present invention further provides a monoclonal antibody derived from a Mouse-Mouse hybridoma CTGF-m21 (FERM ABP-10456) hybridoma.

  In the seventh aspect, the present invention further provides a method for immunizing a non-human animal with an expression vector containing a CTGF-encoding DNA or a functional fragment thereof so as to express the antibody and recognizing the CTGF, or the antibody. There is provided a method for producing the above-described antibody of the present invention, which comprises taking out a producing cell and obtaining a polyclonal or monoclonal antibody recognizing the CTGF.

In that embodiment, the CTGF is human CTGF.
In another embodiment thereof, the non-human mammal is a rodent or ungulate.
In another embodiment thereof, the rodent is a mouse.
In another embodiment thereof, the mouse is a human antibody-producing transgenic mouse.

In another embodiment thereof, the vector can further comprise DNA encoding a secretory signal peptide.
In another embodiment thereof, the vector can further comprise DNA encoding a peptide comprising a localization signal and an immune enhancement signal.

  Preferably, the vector contains a DNA encoding each of a secretion signal peptide, a localization signal peptide, an immune enhancement signal peptide, and a CTGF protein in this order from the 5 ′ end side.

In another embodiment thereof, the cell producing the antibody is a spleen cell, B cell or lymphocyte.
In another embodiment thereof, the ungulate is a cow.
In another embodiment thereof, the body fluid is serum or milk.

In another embodiment thereof, the polyclonal or monoclonal antibody has a binding constant (KA) of 3.0 × 10 ⁷ M ⁻¹ or more and / or a dissociation constant (KD) of 5.6 × 10 ⁻⁹ M or less for the human CTGF. Characterized.

  An example of the functional fragment is DNA encoding module 1, module 2, module 3 or module 4 of human CTGF.

  In another embodiment thereof, the method of the present invention further comprises selecting cells producing the antibody or the hybridoma by flow cytometry.

  According to the present invention, compared with an antibody obtained by a conventional antibody production method for directly immunizing an animal with an antigen, the binding affinity to CTGF having a natural higher-order structure is remarkably high and the biological activity of the CTGF is specific. And highly inhibitory antibodies are provided. The discovery of such antibodies is very surprising to those skilled in the art. When the antibodies of the present invention are produced in vivo, particularly in human antibody-producing transgenic animals, human antibodies are obtained, which are related to CTGF-related diseases in humans, such as fibrosis and cell proliferative diseases. It can be used without rejection for effective treatment. As described above, the present invention can provide an antibody having the above-mentioned novel characteristics that could not be expected to have a sufficient effect with an antibody produced by a conventional antibody production method. Is industrially beneficial and useful for.

Hereinafter, the present invention will be described in more detail.
CTGF
CTGF has been identified in mammals such as humans, mice, rats, pigs and cows, and birds such as chickens, and is secreted from cells and interacts with its specific cell surface receptors, thereby making connective tissue particularly It is one of the growth factors with activities involved in cell growth and extracellular matrix synthesis.

  In the present invention, CTGF is CTGF derived from animals, particularly vertebrates, preferably mammals, more preferably humans. For example, the amino acid and base sequence of CTGF derived from a mammal can be obtained from the GenBank database (NCBI). For example, the CTGF sequence is registered under the accession numbers CAA63267, X92511, P29279, NM001554, NM001901 in humans; NP034347 in mice; NP071602, NM022266 in rats; NP776455, NM174030 in cattle; AAD00174 in pigs.

  Human CTGF is a glycoprotein consisting of 322 amino acids (349 amino acids when including a signal peptide), has 39 rich cysteine residues in addition to human-derived sugar chains, and at least 5 disulfides. It is characterized by the presence of bonds. In addition, as shown in FIG. 1, this protein is composed of four functional domains, namely module 1 which is an insulin-like growth factor binding protein, module 2 which is a von Willebrand type C factor, and thrombospondin type 1 repeat. Module 3 includes module 4, which is a C-terminal cysteine knot-like domain (CTCK).

  The amino acid sequence of human CTGF and the base sequence encoding it are shown as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11 in the sequence listing described below. The amino acid sequences of modules 1 to 4 and the base sequences encoding them are respectively SEQ ID NOs: 3 and 4 for module 1, SEQ ID NOs: 5 and 6 for module 2, SEQ ID NOS: 7 and 8 for module 3, and SEQ ID NO: 7 The numbers 9 and 10 are shown.

  In the present invention, when there is a mutant of CTGF, for example, a mutant that occurs within an individual due to a mutation such as a polymorphism or a splice mutation, such a mutant is also subject to the present invention in the same manner as CTGF. It can be. Such a mutant is composed of one or more, for example several amino acids or nucleotides in the amino acid sequence or base sequence shown by CTGF or a functional fragment (eg module, preferably modules 1-4 of human CTGF). Substitutions, deletions or additions can be included. Here, “several” represents 2 to 10 integers. Alternatively, such a mutant has 80% or more, 90% or more, 95% or more, or 98% or more identity with the amino acid sequence or base sequence. Here,% identity can be readily determined using known programs such as the BLAST program that can take into account the introduction of gaps (S. Karlin et al., Proceedings of the Natinal Academic Sciences USA 90: 5873-5877 (1993)). ).

In the following description, CTGF and its mutants are generically referred to as CTGF.
Production of antibodies by DNA immunization Production of the antibodies of the present invention is accomplished by using DNA immunization techniques.
In DNA immunization, DNA encoding CTGF protein is cloned from tissue or cells of animals such as humans (for example, skin fibroblasts, endothelial cells), and the resulting cDNA is inserted into an animal expression vector. Immunize animals. At this time, an antibody against human CTGF obtained by expression of the DNA in the animal is produced.

  The present invention is characterized in that by using DNA immunization, CTGF is produced in a native state in vivo, and antibodies against it are produced. The native state means adopting the natural higher-order structure (disulfide bond, sugar chain, etc.) of CTGF. Therefore, the antibody produced in the present invention is an antibody against natural CTGF. A preferred antibody is an antibody against native human CTGF.

Cloning of DNA encoding CTGF can be performed as follows.
Based on the cDNA base sequence of CTGF described in the data bank or in the literature, a primer consisting of sequences at the 5 ′ end and 3 ′ end is designed and synthesized. The primer usually has a size of about 15 to 25 bases, and two primers are prepared that can be annealed to the sense strand and the antisense strand of the CTGF. Primer synthesis can be performed using a commercially available DNA automatic synthesizer.

  Total RNA was prepared from animal tissues such as humans or cells (eg, skin fibroblasts, endothelial cells) by the chloroform / phenol method, and poly A (+) RNA (mRNA) was obtained by oligo dT cellulose column chromatography. Obtain and perform cDNA synthesis in the presence of reverse transcriptase. Since cDNA synthesis and cloning kits are commercially available from, for example, Amersham and Invitrogen, it is convenient to use these kits. The cDNA synthesized in this way is used as a template, and a polymerase chain reaction (PCR) is performed using the primers prepared as described above to amplify the cDNA encoding CTGF.

PCR is preferably performed using a commercially available thermal cycler (eg, Perkin Elmer-9600, Eppendorf-Master cycler gradient). As PCR conditions, for example, 10 × PCR buffer (100 mM Tris (pH 8.3), 500 mM KCl, 15 mM MgCl ₂ and 0.1% (W / V) gelatin), dNTPs 2 mM, plumer 2.5 μM to 10 μM, heat resistance such as Taq polymerase DNA polymerase (1 to 2.5 units; rTaq (Takara), Ampli Taq (Promega), etc.) is added to carry out the reaction. PCR may be performed, for example, at 95 ° C, 2 to 5 minutes, followed by denaturation at 95 ° C, 0.2 to 1 minute, annealing at 50 to 65 ° C, 0.5 to 1 minute, and 72 ° C, 0.5 It includes 15 to 40 cycles, with extension at ˜2 minutes as one cycle, and finally extension at 72 ° C. for 5 minutes.

  cDNA cloning and PCR are described in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989), Ausubel et al., Current Protocols in Molecular Biology (1989), and can be referred to.

  The sequence of certain functional fragments of CTGF can be determined based on comparison of identity with CTGF whose function and sequence are already known. The comparison can be performed using the BLAST program described above. Known functional fragments of CTGF are, for example, human CTGF modules 1 to 4. DNA encoding the functional fragment can be synthesized using an automatic DNA synthesizer.

  The DNA encoding CTGF obtained as described above or a functional fragment thereof (for example, human CTGF and its modules 1 to 4) is then inserted into an expression vector.

  The vector can take the form of, for example, a plasmid, virus, phage or the like. Preferred vectors are animal cell expression vectors such as insect cell expression vectors or mammalian cell expression vectors, preferably mammalian cell expression vectors. Vectors are commercially available and can be used in the present invention. Examples of mammalian cell expression vectors include adenovirus vectors, viral vectors such as vaccinia virus vectors, plasmid vectors such as pcDNA3.1 (Invitrogen) and pCI (Promega).

  Expression vectors can contain regulatory sequences such as promoters, enhancers, origins of replication, ribosome binding sites, polyadenylation sites, or terminators. Transcription is initiated by the binding of RNA polymerase to the promoter in the host cell and translated into the target protein or peptide. Examples of preferred promoters are viral promoters such as CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, EBV (Epstein-Barr virus) and the like.

  In the present invention, the vector preferably contains DNA encoding a peptide containing a localization signal and an immune enhancement signal.

  An immunopotentiating signal is any peptide that enhances the immunogenic or antigenic activity of CTGF, as defined above. Such peptides include peptides having immunoadjuvant activity, T cell epitopes, and the like. A T cell epitope is one that can recognize and bind to a T cell receptor. Immunopotentiation signals include, for example, various viral antigens, epitopes of various bacterial toxins, epitopes of T cell-dependent antigens such as keyhole limpet hemocyanin (KLH), ovalbumin (OVA), bovine serum albumin (BSA), etc. including.

  Examples of immune enhancement signals include, but are not limited to, the following (R.S. Chambers and S.A. Johnston, Nature Biotechnology 21 (9): 1088-1092 (2003)).

GlnAlaValHisAlaAlaHisAlaGluIleAsnGlu (SEQ ID NO: 12);
GlnTyrIleLysAlaAsnSerLysPheIleGlyIleThrGluLeu (SEQ ID NO: 13);
PheAsnAsnPheThrValSerPheTrpLeuArgValProLysValSerAlaSerHisLeuGluGlnTyr (SEQ ID NO: 14); and
AsnGlnArgGlyThrGluLeuArgSerProSerValAspLeuAsnLysProGlyArgHis (SEQ ID NO: 15).

In the present invention, one or more immune enhancement signals can be used in combination.
The localization signal is any peptide having a function of transferring CTGF expressed and translated in cells to the cell membrane and anchoring secreted CTGF on the cell membrane. In the present invention, the localization signal is one of the important components and is necessary for the expressed CTGF to remain on the cell surface at a high concentration for a certain period of time so that a high antibody titer CTGF antibody is produced. It is. Examples of localization signals are MetGlyCys and MetGlyCysCys (SEQ ID NO: 16), allowing translocation to the cell membrane via the myristoylated 1-position Gly residue, as well as palmiticated 3- and 4-positions Allows target proteins to be anchored on the cell membrane via the Cys residues (Zuber, MX, Strittmater, SM and Fishman, MC (1989) Nature 341, 345-348; Skene, JHP and Virag, I. (1989) J. Cell Biol. 108, 613-624; Liu, Y. et al. (1993) Biochemistry 72, 10714-10719). Localization signals that can be used in the present invention are not limited to the above examples, and any signals can be used as long as they have the same anchoring action.

  The localization signal and the immune enhancement signal are preferably linked in this order from the N-terminal side. A linker consisting of one to several arbitrary amino acids may be bound between these two signals. In addition, CTGF or a functional fragment thereof is linked to the C-terminal side of a peptide containing a localization signal and an immune enhancement signal, optionally via a linker consisting of one to several arbitrary amino acids. .

  It is preferable that the vector further links DNA encoding a secretory signal peptide for transporting CTGF expressed in the cell or a functional fragment thereof to the cell membrane. A suitable example of such a signal peptide is a signal peptide of CTGF, for example, a signal peptide of human CTGF (sequences at positions 1 to 27 of SEQ ID NO: 1).

  In the present invention, a preferred vector is a fusion protein containing a secretion signal peptide, a localization signal peptide, an immune enhancement signal peptide, and CTGF in this order from the N-terminal side in the order downstream from the promoter. Contains the encoding DNA. The fusion protein expressed in the cell moves to the cell membrane, is cleaved from the signal peptide and secreted, and remains as an anchor in the cell membrane. As a result, an antibody against CTGF is easily generated.

  The vector further contains a base sequence encoding a tag for detecting the expression of the target DNA, such as a FLAG tag, a histidine tag (for example, His6 to His10), if necessary, at the 5 ′ end or 3 of the target DNA. 'Can be joined to the end. In particular, a fluorescent tag such as a FLAG tag is useful for detecting the expression of the target protein.

  An expression vector containing DNA encoding CTGF or a functional fragment thereof is injected into an animal body part, for example, subcutaneously. The infusion can occur several times, about every 1-2 weeks, for example, over about 1-2 months. The injection amount of the vector is, for example, 17.5 μg antigen / time.

  Animals refer to vertebrates (fish, reptiles, amphibians, birds, mammals), with preferred animals being birds and non-human mammals. More preferred animals are non-human mammals such as rodents (eg mice, rats, hamsters, guinea pigs, etc.), ungulates (eg cows, horses, sheep, pigs, goats etc.), rabbits and the like.

  Since the present invention aims to produce antibodies, animals (except humans) capable of producing human antibodies or humanized antibodies are preferably used. Such animals are human antibody-producing transgenic animals, and are known, for example, from mice and cows (Japanese Patent Publication No. 4-504365, Japanese Patent Publication No. 6-500233, US Pat. No. 5,545,806, US Pat. No. 5,569,825, LL Green, J. Immunol. Methods 231: 11-23 (1999)). In particular, KM mice (Kirin Brewery), Xenomouse (Abjenix) and the like are known as transgenic mice producing human antibodies. In these mice, all or part of the original antibody gene of the mouse is replaced with a human-derived antibody gene, whereby the mouse can produce a human antibody or a humanized antibody. That is, in the human antibody, both the variable region and the constant region of the immunoglobulin heavy chain and light chain are derived from a human immunoglobulin gene. A humanized antibody (or chimeric antibody) is a part of a variable region and constant region of a heavy chain or light chain derived from a human immunoglobulin gene, preferably a part of CDR of a hypervariable region or a human antibody molecule. All replaced by part of the corresponding heavy or light chain variable and constant regions derived from immunoglobulin genes of non-human animals such as mice, preferably part or all of the CDRs of the hypervariable region Such a chimeric immunoglobulin.

  As for the production of a humanized antibody, for example, there is a technique for producing a humanized antibody without using an animal. That is, humanized antibodies can be obtained, for example, by chain shuffling or using phage display technology. For example, a polypeptide comprising a heavy or light chain variable domain of a non-human antibody specific for CTGF is conjugated to a repertoire of human complement (light or heavy) chain variable domains. A hybrid pairing specific for the antigen of interest is selected. The human chain from the selected pairing can then be combined with a repertoire of human complement variable domains (heavy or light) and humanized antibody polypeptide dimers can be selected for binding specificity for the antigen. Techniques described for the production of humanized antibodies that can be used in the methods of the invention are disclosed, for example, in US Pat. Nos. 5,565,332, 5,585,089, 5,694,761, and 5,693,762.

  In animals immunized by DNA immunization according to the present invention, production of antibodies against transiently expressed CTGF or functional fragments thereof is induced. After production of the antibody, the desired antibody is collected from animal body fluids such as serum, plasma, milk and the like. The antibody obtained here is a polyclonal antibody. The antibody isotype may be any of IgG, IgM, IgA, IgD, IgE and the like. The antibody class is IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc., and the light chain is κ chain, λ chain, or the like. The same kind of antibody is used for monoclonal antibodies.

The present invention further includes the above-mentioned antibody or the following monoclonal antibody fragment. Examples of such fragments include Fc, Fd, Fab, Fab ′, (Fab ′) _{2 and the} like. For example, fragments can be obtained by degradation with a protease such as papain, trypsin, pepsin.

  The monoclonal antibody of the present invention can be prepared by a general method for producing a monoclonal antibody as follows.

  Spleen cells, B cells or lymphocytes (or lymph nodes) are removed from animals (excluding humans) immunized by the above DNA immunization method, preferably mice, more preferably human antibody-producing transgenic mice, and these cells are extracted. And myeloma cells (also called myeloma cells), preferably mouse myeloma cells, are fused to produce a hybridoma. For cell fusion, a cell-aggregating medium such as polyethylene glycol is used.

  From the resulting hybridoma, an antibody that specifically recognizes natural CTGF, preferably the natural higher-order structure of CTGF, is selected. For the selection, flow cytometry can be preferably used. For detection, fluorescently labeled secondary antibodies that specifically bind to human or mouse immunoglobulins can be used.

  The established hybridoma is injected intraperitoneally into a nude mouse, ascites is collected, and the ascites is passed through a protein A or protein G sepharose column, and the target monoclonal antibody with high purity can be recovered.

  For methods of producing monoclonal antibodies, see Kohler et al., Nature, 256: 495 (1975), Ausubel et al., Current Protocols in Molecular Biology (1989), Kozbor et al., Immunology Today 4:72 (1983), Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96 (1985), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 8, 1988, and the like, which can be referred to for carrying out the present invention.

  Therefore, according to the present invention, an antibody that recognizes CTGF by immunizing a non-human animal with an expression vector capable of expressing DNA encoding CTGF or a functional fragment thereof (for example, DNA encoding a module constituting CTGF) is provided. There is provided a method for producing the above-mentioned antibody, which comprises removing a body fluid containing the cell or a cell producing the antibody, and obtaining a polyclonal or monoclonal antibody recognizing the CTGF.

According to an embodiment of the present invention, the non-human animal is preferably a mammal, more preferably a rodent or ungulate. An example of a rodent is a mouse, preferably a human antibody-producing transgenic mouse. The ungulates are preferably bovine, goat, sheep, etc., preferably human antibody-producing transgenic cattle.
In the above method, the body fluid is, for example, serum or milk.

  According to an embodiment of the present invention, the cell producing the antibody is a spleen cell, a B cell or a lymphocyte. As described above, these cells are fused with myeloma cells to form hybridomas, which are used to produce the monoclonal antibodies of the present invention.

  Furthermore, according to an embodiment of the present invention, the method of the present invention further comprises selecting cells producing the antibody or the hybridoma by flow cytometry. For detection, fluorescently labeled secondary antibodies that specifically bind to human or mouse immunoglobulins can be used. From the antibodies obtained by this selection method, harmful antibodies that cause side effects on humans are excluded.

  Anti-module antibodies can also be obtained by selecting from a group of anti-CTGF polyclonal or monoclonal antibodies induced in vivo by full-length CTGF gene expression by DNA immunization using a selection technique such as immunoabsorption or flow cytometry. be able to.

Antibody Features According to the present invention, an antibody that specifically recognizes CTGF or its functional fragment (eg, module) having a natural higher-order structure by the DNA immunization method as described above (ie, polyclonal antibody or monoclonal antibody) Is obtained. In particular, when DNA immunization is performed on a human antibody-producing transgenic animal (eg, mouse, bovine), a human antibody or a humanized antibody that specifically recognizes human CTGF retaining its higher-order structure or each module thereof Can be obtained.

  As such an antibody, for example, a monoclonal antibody against each of modules 1 and 2 of human CTGF, that is, a monoclonal antibody derived from Mouse-Mouse hybridoma CTGF-m11 (FERM ABP-10454), Mouse-Mouse hybridoma CTGF-m12 (FERM ABP -10455) and Mouse-Mouse hybridoma CTGF-m21 (FERM ABP-10456) -derived monoclonal antibodies and the like. However, the present invention is not limited to these particular antibodies. For example, human antibody-producing transgenic animals are used to produce human antibodies against human CTGF or each of modules 1 to 4, and similarly, It is also possible to obtain a target human antibody.

The antibody of the present invention has a binding constant for natural CTGF of 3.0 × 10 ⁷ M-1 or more, 5.0 × 10 ⁷ M-1 or more, preferably 1.0 × 10 5 when measured by the surface plasmon resonance method of BIAcore (registered trademark). ⁸ M ^-1 or more, 1.8 x 10 ⁸ M ^-1 or more, 3.0 x 10 ⁸ M ^-1 or more, more preferably 5.0 x 10 ⁸ M ^-1 or more, particularly preferably 1.0 x 10 ⁹ M ^-1 or more, and / or Alternatively, the dissociation constant for natural CTGF is 5.6 × 10 ⁻⁹ M or less, preferably 3.5 × 10 ⁻⁹ M or less, 3.0 × 10 ⁻⁹ M or less, more preferably 2,5 × 10 ⁻⁹ M or less, 2,0 × 10 ^-9 M or less, particularly preferably 1.0 × 10 ^-9 M or less, 5.0 × 10 ^-10 M or less.

  The antibody of the present invention can specifically recognize the natural higher-order structure of CTGF, preferably human CTGF. Here, specific means that CTGF having a natural higher-order structure is recognized but the modified CTGF is not recognized, or CTGF having a natural higher-order structure is more easily recognized than the modified CTGF. Means that. Such a relatively high recognition degree (or immunological reactivity), in other words, a binding ability that is 10 times or more, preferably 100 times or more, more preferably 1,000 times or more is specifically mentioned in the present specification. Recognize. The high binding affinity for native human CTGF that has not been achieved by conventional immunization methods makes it possible to significantly inhibit or suppress the biological activity of human CTGF at a practical level in vivo. Here, the biological activity includes proliferation of fibrous connective tissue, enhanced cell proliferation including angiogenesis, and the like.

Pharmaceutical Composition The present invention further provides a pharmaceutical composition comprising an effective amount of the above-described antibody of the present invention.
The antibody used in the pharmaceutical composition of the present invention is preferably an antibody against human CTGF or a functional fragment thereof (for example, modules 1 to 4), preferably a human antibody or a humanized antibody, particularly preferably a human antibody. It is. The antibody may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.

  By flow cytometry, a hybridoma that produces a monoclonal antibody that effectively acts only on diseased cells or tissues without substantially adversely affecting normal cells or tissues can be selected.

  The composition of the present invention can be used for the treatment or prevention of fibrosis or sclerosis associated with fibrosis, or cell proliferative disease.

  Examples of the sclerosis associated with fibrosis or fibrosis include fibrosis of organs such as kidney, lung, liver, eyeball, heart, scleroderma, interstitial fibrosis, epidural fibrosis and the like. Abnormal expression of CTGF is associated with general tissue scarring, tumor-like growth of the skin, and persistent scarring of blood vessels, leading to poor blood carrying capacity, hypertension, hypertrophy, etc. Other diseases related to CTGF include cancers including cutaneous fibroma, symptoms related to abnormal expression of endothelial cells, breast cancer fibrosis, angiolipoma, angioleiomyoma, atherosclerosis, systemic Sclerosis (eg, atherosclerotic plaque, inflammatory bowel disease, Crohn's disease, angiogenesis), arthritis and other disease symptoms, angiogenesis involving glaucoma, inflammation (eg, arthritis), tumor growth metastasis, interstitial disease , Skin diseases, arthritis (such as rheumatoid arthritis), arteriosclerosis, diabetes (such as diabetic neuropathy), hypertension, other renal disorders, surgery, chemotherapy, radiation treatment, allograft rejection, Various diseases, disorders or symptoms such as transplant rejection are included.

  Fibrosis refers to a state in which fibrous connective tissue has proliferated in the organ or organ as described above, causing tissue hardening and atrophy, and destruction and devastation of a normal organ or organ structure. Fibrosis can be caused by various injuries or diseases, but is often caused by chronic transplant rejection associated with transplantation of various organs. Fibrosis generally involves abnormal production, accumulation or deposition of cellular stromal matrix components, causing, for example, overproduction and increased deposition of collagen and fibronectin.

  The cell proliferative disorder is an abnormally proliferative cancer. CTGF has an effect of enhancing angiogenesis and is said to be involved in angiogenesis particularly in malignant tumors (that is, cancer) (Patent Document 4). Examples of cancer include acute lymphoblastic leukemia, dermal fibroma, breast cancer, breast cancer gland dysplasia, angiolipoma, angioleiomyoma, adhesion cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer , Liver cancer, other cancers including tumor growth and metastasis, and the like.

  Treatment of various diseases or disorders related to CTGF such as fibrosis or sclerosis associated with fibrosis, angiogenesis, etc., if CTGF can inhibit or suppress the binding of its cell surface receptors and transmission of proliferation signals Or it may be useful for prevention. One inhibitor or suppressor for this is an antibody against human CTGF or its surface antigen epitope.

  According to an embodiment of the present invention, a therapeutic agent can be bound to the antibody. The antibody delivers the drug to or near the target disease site where CTGF is present and inhibits or suppresses the function of CTGF, while the drug cures, reduces or ameliorates the symptoms of the disease. Such a form of medicine is a so-called molecular targeted drug.

  Drugs include all drugs that are or are being used as therapeutic drugs for the above diseases. Such agents are, for example, synthetic or natural, low or high molecular weight, proteinaceous or nonproteinaceous, or nucleic acid or nucleotide substances. For example, examples of therapeutic agents for fibrosis include prostaglandin E1, steroids, PPAR ligands, angiotensin II type I receptor antagonists, soluble TGF type II receptors, HGF, etc., and examples of anticancer agents include alkylating agents. For example, ifosfamide, nimustine, cyclophosphamide, dacarbazine, ranimustine, etc., antimetabolite such as gemcitabine, fluorouracil, etc., anticancer antibiotics such as daunorubicin, doxorubicin, bleomycin, mitomycin C, etc. And platinum complexes such as vinblastine, such as cisplatin.

  The binding between the antibody and the drug is preferably performed via a linker. The linker includes, for example, a substituted or unsubstituted aliphatic alkylene chain, and a group capable of binding to a functional group of an antibody or a drug at both ends, such as an N-hydroxysuccinimide group, an ester group, a thiol group, and an imide carbonate. Group, aldehyde group, etc. (Introduction to antibody engineering, Jinjinshokan, 1994).

  If necessary, it can be encapsulated in liposomes in order to facilitate delivery of the drug into the cell. Preferred liposomes are positively charged liposomes, positively charged cholesterol, membrane-permeable peptide-bonded liposomes, etc. (Nakanishi Mamoru et al., Protein Nucleic Acid Enzyme 44: 1590-1596 (1999), Futaki Shiro, Chemistry and Biology 43: 649-653 ( 2005), Clinical Cancer research 59: 4325-4333 (1999)).

Administration is parenteral, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal and the like.
Preparations for parenteral administration include solutions, suspensions, emulsions, and the like in aqueous solutions such as sterilized physiological saline solution, buffered salt solution, and organic solvent-containing solution. Examples of the organic solvent include ethanol, propylene glycol, polyethylene glycol, vegetable oil, fatty acid esters such as ethyl oleate, and the like. Moreover, additives, such as a nutrient, an electrolyte solution, a preservative, and an antioxidant, may be contained in the preparation.

  The amount of the active ingredient of the antibody is 100 to 2,500 μg / ml per dose, or 1.0 to 10 mg per kg of human adult patient body weight, but is not limited thereto.

  The administration frequency is, for example, several times at a frequency of once every 1 to 2 weeks or once every 2 to 3 weeks for about 2 months.

Diagnostic composition The present invention further provides a diagnostic composition comprising the antibody.
The composition of the present invention is used for diagnosis of, for example, fibrosis or sclerosis associated with fibrosis, or cell proliferative disease, but is not limited thereto, for detection of other diseases and disorders described above. Can also be used. In such diseases, CTGF expression is in a state higher than a normal value or a standard value, resulting in abnormal growth of connective tissue, tissue hardening and atrophy, and destruction of an organ or organ structure. For this purpose, the above-mentioned diseases can be diagnosed by measuring the presence or amount of CTGF using the antibody of the present invention. As the antibody, not only an antibody against human CTGF but also an antibody against CTGF of animals other than humans, preferably mammals, can be used. CTGF has high sequence identity between mammals, for example, human and mouse have 93.5% identity at the amino acid level.

  Examples of assays include conventional general assays, such as enzyme-linked immunosorbent assays (ELISA), fluorescent antibody assays, radioimmunoassays, radioimmunoprecipitation, dot blot assays, inhibition or competition assays, sandwich assays, latex beads Examples include but are not limited to agglutination assays.

  The antibody of the present invention is characterized by specifically recognizing native CTGF (preferably human CTGF), particularly specific to a continuous or discontinuous cell surface antigen epitope of the natural conformation of CTGF. Can be recognized.

  Monoclonal antibodies are preferred in order to achieve high specificity. For the purpose of diagnosing or detecting a disease, the antibody need not be a human antibody or a humanized antibody, and may be, for example, a mouse monoclonal antibody. Examples of such antibodies include, but are not limited to, Mouse-Mouse hybridoma CTGF-m11 (FERM ABP-10454), Mouse-Mouse hybridoma CTGF-m12 (FERM ABP-10455), Mouse-Mouse hybridoma CTGF- It is a monoclonal antibody derived from a hybridoma of m31, Mouse-Mouse hybridoma CTGF-m32, Mouse-Mouse hybridoma CTGF-m33, or Mouse-Mouse hybridoma CTGF-m41 (see Examples below).

  Fibrosis detectable by the method of the present invention or sclerosis associated with fibrosis is, for example, fibrosis such as renal fibrosis, pulmonary fibrosis, liver fibrosis, or dermal fibrosis (also referred to as scleroderma), and The cell proliferative disease is, for example, cancer, but is not limited thereto.

  The sample is a biological sample, such as a tissue or cell biopsy of the disease site, blood, serum, plasma, lymph, body fluid such as urine, and the like. These samples are homogenized, centrifuged, or diluted as necessary, the antibody of the present invention is added in an appropriate buffer, and the complex of human CTGF as an antigen and the antibody is assayed as described above. Detect by method. In this case, the antibody of the present invention may be labeled, or a labeled secondary antibody can be used.

Labels include enzymes (eg, horseradish peroxidase, alkaline phosphatase, etc.), radioisotopes (eg, ³² P, ³⁵ S, ³ H, ¹²⁵ I, etc.), fluorescent substances (eg, rhodamine, fluorescamine, dansyl chloride, derivatives thereof, etc. ) Etc.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

(1) Construction of expression vector Full-length gene (SEQ ID NO: 2) of human CTGF or each of module 1 (SEQ ID NO: 4), module 2 (SEQ ID NO: 6), module 3 (SEQ ID NO: 8), and module Each gene fragment of 4 (SEQ ID NO: 10) was incorporated into a mammalian expression vector (VV8; Invitrogen) with a FLAG tag. Expression vectors also include cytomegalovirus (CMV) promoter, localization signal sequence (MetGlyCys) and immune enhancement signal sequence (chicken OVA epitope; RS Chambers and SA Johnston, Nature Biotechnology 21 (9): 1088-1092 (2003)) DNA encoding a peptide consisting of

  In order to obtain a monoclonal antibody that recognizes the higher-order structure of human CTGF and inhibits its biophysiological activity, vv8 / CTGF, which is a construct containing the full-length human CTGF gene, is the optimal antigen gene for immunization. A construct into which any of module 1, module 2, module 3, and module 4 is inserted can also be used as an immunizing antigen gene.

Immunize whether the constructed gene construct (ie vv8 / human CTGF full-length gene, vv8 / module 1, vv8 / module 2, vv8 / module 3 and vv8 / module 4) is expressed on the cell surface as designed Previously, it was verified using mammalian cells derived from human kidney (HEK293T). That is, the constructed gene construct was transiently introduced into mammalian cells. The introduced mammalian cells were cultured in a D-MEM medium supplemented with 10% FCS in a 5% CO ₂ incubator for 24 hours at 37 ° C. and used for flow cytometry (FCM) analysis. When FCM analysis was performed, a FLAG M2 antibody (Sigma-ardrich) against the tag added to the transgene was added to the cultured cell suspension into which the gene was introduced, and allowed to stand for 30 minutes. Thereafter, a fluorescent-labeled anti-mouse Ig antibody (Beckmancoulter) that specifically recognizes the primary antibody was added to the reaction solution, left to stand for 30 minutes, and used for FCM analysis.

  As a result, it was proved that all five gene constructs constructed according to the present invention were expressed on the cell surface.

(2) Production of antibodies by DNA immunization
In the DNA immunization method, an animal (mouse or human) can be obtained by using any of various gene transfer methods (for example, intramuscular injection, electroporation, gene gun, etc.) with respect to an immunized animal by singly or mixing the above gene constructs. Antibody-injecting transgenic mice) were injected subcutaneously into cells.

  An animal producing a strong specific antibody that recognizes human CTGF full-length gene-transfected cells was dissected, and B cells were isolated according to a standard method to produce a monoclonal antibody. Specifically, it was performed as follows.

Hybridomas were prepared by fusing immune cells obtained from the spleen or lymph nodes of immunized Balb / c mice with myeloma cells. Myeloma cells are cell lines derived from mice, 8-azaguanine resistant mice (derived from BALB / c) myeloma cell line P3-X63Ag8-U1 (P3-U1) [Eur. J. Immunol., 6 , 511 (1976)], SP2 / 0-Ag14 (SP-2) [Nature, 276 , 269 (1978)], P3-X63-Ag8653 (653) [J. Immunol., 123 , 1548 (1979)], P3-X63-Ag8 (X63) [Nature, 256 , 495 (1975)], etc. Any myeloma cells that can proliferate in vitro But you can. For culturing and subculture of these cell lines, according to a known method [Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 8, 1988]; × 10 ⁷ or more cells were secured.

After washing the antibody-producing cells and myeloma cells obtained from the immunized mouse, a cell aggregating medium such as polyethylene glycol-1000 (PEG-1000) was added, the cells were fused, and suspended in the medium. . For washing the cells, MEM medium or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g NaCl, 1 liter distilled water, pH 7.2) or the like was used. As a medium for suspending fused cells, HAT medium [glutamine (4 mM), 15% fetal calf serum (FCS), hypoxanthine (10 ⁻⁴ M) is used so that only the desired fused cells can be selectively obtained. ), Thymidine (1.5 × 10 ⁻⁵ M) and aminopterin (4 × 10 ⁻⁷ M)].

  After the culture, a part of the culture supernatant was taken, and a sample that reacted with the antigen protein and did not react with the non-antigen protein was selected by enzyme immunoassay (CELISA) using forced expression cells and FCM. Subsequently, cloning was performed by a limiting dilution method, and a stable and high antibody titer was selected as a monoclonal antibody-producing hybridoma strain.

  The enzyme immunoassay using forced expression was performed as follows. Cells in which the antigen protein was forcibly expressed were coated on a 96-well plate, and the hybridoma culture supernatant was reacted as the first antibody. After the first antibody reaction, the plate was washed and the second antibody was added. Here, the second antibody is an antibody that can recognize mouse immunoglobulin of the first antibody, and is an antibody labeled with horseradish peroxidase (HRP). After the reaction, a fluorescent substrate corresponding to the enzyme labeled with the second antibody was added and analyzed with a fluorescence measuring plate reader.

Next, large-scale preparation of monoclonal antibodies from ascites was performed as follows. Balb / c mice pretreated with 0.5 ml pristane were injected intraperitoneally with 1-3 × 10 ⁶ cloned hybridoma cells in 0.5 ml phosphate buffered saline, pH 7.4. Approximately 2 weeks later, ascites was collected and monoclonal antibodies were affinity purified with protein A or protein G.

  In order to select a hybridoma cell line that produces a monoclonal antibody that recognizes the natural higher-order structure of human CTGF from a number of hybridoma cell lines established by DNA immunization, the FCM analysis system is used in the same manner as in (1) above. Using.

The monoclonal antibody prepared by the above technique is as follows.
Mouse-Mouse hybridoma clone 30D2 from CTGF-m11 hybridoma;
Mouse-Mouse hybridoma clone 30E9 from CTGF-m12 hybridoma;
Mouse-Mouse hybridoma clone 1 from CTGF-m21 hybridoma;
Mouse-Mouse hybridoma clone 22D10 from CTGF-m31 hybridoma;
Mouse-mouse hybridoma clone 21C12 from CTGF-m32 hybridoma;
Mouse-Mouse hybridoma clone 21H12 from CTGF-m33 hybridoma;
Mouse-Mouse hybridoma clone 8E7 derived from CTGF-m41 hybridoma.

  clone 30D2 and clone 30E9 are monoclonal antibodies (MAb) that specifically recognize human CTGF module 1, clone 1 is a MAb that specifically recognizes human CTGF module 2, clone 22D10, clone 21C12 and clone 21H12 are human CTGF module 3 Clone 8E7 that specifically recognizes human CTGF module 4 is a MAb that specifically recognizes human CTGF module 4.

  Among the prepared hybridomas, Mouse-Mouse hybridoma CTGF-m11 (FERM ABP-10454), Mouse-Mouse hybridoma CTGF-m12 (FERM ABP-10455) and Mouse-Mouse hybridoma CTGF-m21 (FERM ABP-10456) The hybridoma was deposited on November 25, 2005 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Chuo 6-1, 1-chome East, Tsukuba, Ibaraki, Japan).

  Furthermore, in the above method, a natural human CTGF, its module 1, module 2, module 3, and module 4 are identified by obtaining a human antibody-producing transgenic mouse instead of a normal mouse and using it experimentally. A fully human antibody that can be recognized automatically.

(3) Antibody characterization analysis Purified immunoglobulins of the prepared hybridoma For detailed analysis of immunoglobulin, epitope analysis, surface plasmon resonance (SPR) analysis, in vivo skin fibrosis model (mouse fibrosis model; T. Mori) Et al., J. Cellular Physiology 18: 153-159 (1999)).

For epitope analysis, mammalian cells into which any of the module 1, module 2, module 3, and module 4 genes are introduced are cultured in a 5% CO ₂ incubator for 24 hours in the same manner as in (1) above, and FCM analysis is performed in the same manner. (FIGS. 2 to 9). As is apparent from the figure, a high affinity monoclonal antibody (IgG1) that specifically recognizes each module was obtained even at 5,000-fold or 10,000-fold dilution.

  In SPR, each purified immunoglobulin was immobilized on the surface of a Biacore CM5 chip. Purified recombinant human CTGF antigen (CTGF with a natural higher-order structure prepared from mammalian cells HEK293T by a conventional method) was passed through the flow path of a CM5 chip on which various immunoglobulins were immobilized. With this measurement system, the dissociation constant and the binding constant between the non-denatured human CTGF protein and the monoclonal antibody prepared in the present invention were measured.

The measurement conditions are as follows.
Measuring instrument: Biacore 2000 (Biacore);
Ligand immobilized chip: CM5 sensor chip;
Ligand (antibody) dilution buffer: 10 mM acetate buffer (pH 5.0) (with this buffer the ligand was adjusted to a final concentration of 25 μg / ml);
Running buffer: HBS-EP buffer (10 mM HEPES, pH 7.5, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20 (Tween 20), pH 7.4).
The results are shown in Table 1.

As is clear from Table 1 above, the antibody (MAb) of the present invention has a binding constant and a dissociation constant of 1.0 × 10 ⁸ M ⁻ , respectively, using recombinant human CTGF prepared from transformed mammalian cells HEK293T as an antigen. ^{It was 1} or more and 5.6 × 10 ⁻⁹ M or less, indicating a very high affinity.

Note that when the binding constant and dissociation constant of the antibody of the present invention were measured using recombinant human CTGF produced in general-purpose insect (silkworm) cells as an antigen, module 1, module 1 2, The binding constant of module 3 and module 4 is 3.0-8.0 × 10 ⁷ M-1 and the dissociation constant is 1.0-3.2 × 10 ⁻⁸ M. Compared with recombinant human CTGF prepared in mammalian cells Showed a tendency to decrease.

  Furthermore, the biological activity of the antibody of the present invention was examined. Specifically, using a dermatofibrosis model, the inhibitory effect of the disease of the present invention was tested in vivo.

  Fibrosis was induced in the subcutaneous tissue by subcutaneously injecting 800 ng of TGFβ3 for 3 days into Balb / c mice (7 days after birth) and then injecting 400 ng of CTGF for 4 days.

  4 μg of the antibody of the present invention (anti-module 2 antibody) was mixed with CTGF and immediately injected subcutaneously. In the control group, mouse IgG was injected subcutaneously in the same amount.

  The human CTGF antibody obtained in the present invention showed a significant inhibitory effect on in vivo dermal fibrosis in mice as compared with the control group administered with immunoglobulin (negative control) obtained from non-immunized mice (FIGS. 10 and 10). 11). That is, FIG. 10 shows a control plot, and represents a tissue in which a river-like portion that vertically cuts the center of the figure is fibrotic. As shown in FIG. 11, in the test group treated with the antibody of the present invention, the fibrotic portion was greatly reduced to about 1/3 as compared with the control group. From this, it was proved that the antibody of the present invention is effective for suppressing fibrosis.

(Comparative Example 1)
Human CTGF was expressed using insect cells infected with recombinant baculovirus, and recombinant human CTGF purified by affinity column was prepared. As a result of immunizing mice and chickens with this recombinant CTGF, no antibody against human CTGF was produced.

(Comparative Example 2)
Antibody production in mice by DNA immunization was attempted using a VV6 expression vector derived from pcDNA3.1 plasmid (Invitrogen) which can express DNA encoding human CTGF. As a result of antiserum analysis of mice by FCS, the antibody titer of CTGF antibody was found to be quite low.

  According to the present invention, antibodies (including human antibodies) that specifically recognize natural human CTGF having a special structure and adhesiveness, which were difficult to produce with conventional methods, could be easily prepared. Thus, the antibody of the present invention is useful for the treatment or diagnosis of fibrosis or cell proliferative disease related to CTGF.

The basic structure of human CTGF is shown. The FCM analysis result of a mouse polyclonal anti-CTGF polyclonal antibody (antigen: full-length CTGF) is shown. The FCM analysis result of monoclonal antibody clone 30D2 derived from Mouse-Mouse hybridoma CTGF-m11 is shown. The FCM analysis result of monoclonal antibody clone 30E9 derived from Mouse-Mouse hybridoma CTGF-m12 is shown. The FCM analysis result of monoclonal antibody clone 1 derived from Mouse-Mouse hybridoma CTGF-m21 is shown. The FCM analysis result of monoclonal antibody clone 22D10 derived from Mouse-Mouse hybridoma CTGF-m31 is shown. The FCM analysis result of monoclonal antibody clone 21C12 derived from Mouse-Mouse hybridoma CTGF-m32 is shown. The FCM analysis result of monoclonal antibody clone 21H12 derived from Mouse-Mouse hybridoma CTGF-m33 is shown. The FCM analysis result of monoclonal antibody clone 8E7 derived from Mouse-Mouse hybridoma CTGF-m41 is shown. The control group which administered the immunoglobulin (negative control) derived from a non-immune mouse | mouth in the inhibitory effect test with respect to the in vivo skin fibrosis of a Balb / c mouse (7 days after birth) is shown. The test plot which administered the antibody of this invention in the inhibitory effect test with respect to the in vivo skin fibrosis of a Balb / c mouse (7 days after birth) is shown.

Claims (45)

  An antibody produced by DNA immunization or a fragment thereof capable of specifically recognizing connective tissue growth factor (CTGF).   The antibody or fragment thereof according to claim 1, wherein the CTGF is human CTGF.   The antibody or fragment thereof according to claim 1 or 2, wherein the antibody is a human antibody or a humanized antibody.   The antibody or fragment thereof according to any one of claims 1 to 3, which can specifically recognize a natural higher-order structure of the CTGF molecule.   The antibody according to any one of claims 1 to 4, wherein the DNA immunization comprises immunizing a non-human animal with an expression vector that can express the DNA encoding CTGF or a functional fragment thereof. fragment.   The antibody or fragment thereof according to claim 5, wherein the vector further comprises DNA encoding a secretory signal peptide.   The antibody or fragment thereof according to claim 5 or 6, wherein the vector further comprises DNA encoding a peptide comprising a localization signal and an immune enhancement signal.   The antibody or fragment thereof according to any one of claims 5 to 7, wherein the functional fragment is DNA encoding the CTGF module.   The antibody or fragment thereof according to any one of claims 5 to 8, wherein the DNA encoding CTGF or a functional fragment thereof comprises a base sequence represented by SEQ ID NO: 2, 4, 6, 8 or 10.   The antibody or fragment thereof according to claim 5, wherein the animal is a mammal.   11. The antibody or fragment thereof according to claim 10, wherein the mammal is a mouse.   12. The antibody or fragment thereof according to claim 11, wherein the mouse is a human antibody-producing transgenic mouse.   The antibody or fragment thereof according to any one of claims 1 to 12, which can specifically recognize any one of module 1, module 2, module 3 or module 4 of the human CTGF. 14. The antibody according to claim 1, wherein the antibody has a binding constant (KA) of 3.0 × 10 ⁷ M ⁻¹ or more and / or a dissociation constant (KD) of 5.6 × 10 ⁻⁹ M or less to the CTGF. An antibody or fragment thereof.   A pharmaceutical composition comprising the antibody or fragment thereof according to any one of claims 1 to 14.   16. The pharmaceutical composition according to claim 15, wherein the antibody is a monoclonal antibody.   17. The pharmaceutical composition according to claim 15 or 16, wherein the antibody is a human antibody or a humanized antibody.   18. A pharmaceutical composition according to any one of claims 15 to 17, wherein the antibody is conjugated to a therapeutic agent.   The pharmaceutical composition according to any one of claims 15 to 18, which is used for treatment or prevention of fibrosis or sclerosis associated with fibrosis, or a cell proliferative disease.   20. The pharmaceutical composition according to claim 19, wherein the fibrosis or sclerosis associated with fibrosis is renal fibrosis, pulmonary fibrosis, liver fibrosis or scleroderma.   20. The pharmaceutical composition according to claim 19, wherein the cell proliferative disorder is cancer.   A diagnostic composition comprising the antibody or fragment thereof according to any one of claims 1 to 14.   23. The diagnostic composition according to claim 22, wherein the antibody is a monoclonal antibody.   24. The diagnostic composition according to claim 22 or 23, which is used for diagnosis of fibrosis or sclerosis associated with fibrosis, or a cell proliferative disease.   25. The diagnostic composition according to claim 24, wherein the fibrosis or sclerosis associated with fibrosis is renal fibrosis, pulmonary fibrosis, liver fibrosis or scleroderma.   25. The diagnostic composition according to claim 24, wherein the cell proliferative disease is cancer.   The antibody is a monoclonal derived from a hybridoma of Mouse-Mouse hybridoma CTGF-m11 (FERM ABP-10454), Mouse-Mouse hybridoma CTGF-m12 (FERM ABP-10455) or Mouse-Mouse hybridoma CTGF-m21 (FERM ABP-10456). 27. The diagnostic composition according to any one of claims 22 to 26, which is an antibody.   Mouse-Mouse hybridoma Monoclonal antibody derived from CTGF-m11 (FERM ABP-10454) hybridoma.   Mouse-Mouse hybridoma CTGF-m12 (FERM ABP-10455) hybridoma-derived monoclonal antibody.   Mouse-Mouse hybridoma CTGF-m21 (FERM ABP-10456) hybridoma-derived monoclonal antibody.   Immunize a non-human animal with an expression vector that can express the CTGF-encoding DNA or functional fragment thereof, extract a body fluid containing the antibody that recognizes the CTGF, or a cell that produces the antibody, and recognize the CTGF 15. A method for producing an antibody or a fragment thereof according to any one of claims 1 to 14, comprising obtaining a polyclonal or monoclonal antibody.   32. The method of claim 31, wherein the CTGF is human CTGF.   32. The method of claim 31, wherein the non-human animal is a mammal.   34. The method of claim 33, wherein the mammal is a rodent or ungulate.   35. The method of claim 34, wherein the rodent is a mouse.   36. The method of claim 35, wherein the mouse is a human antibody-producing transgenic mouse.   37. The method of any one of claims 31 to 36, wherein the vector further comprises DNA encoding a secretory signal peptide.   38. The method of any one of claims 31 to 37, wherein the vector further comprises DNA encoding a peptide comprising a localization signal and an immune enhancement signal.   32. The method of claim 31, wherein the antibody producing cell is a spleen cell, B cell or lymphocyte.   35. The method of claim 34, wherein the ungulate is a cow.   32. The method of claim 31, wherein the body fluid is serum or milk.   42. The method of any one of claims 31 to 41, further comprising fusing the antibody producing cell with a myeloma cell to form a hybridoma. 43. Any one of claims 31 to 42, wherein the polyclonal or monoclonal antibody has a binding constant (KA) of 3.0 × 10 ⁷ M ⁻¹ or more and / or a dissociation constant (KD) of 5.6 × 10 ⁻⁹ M or less to the CTGF. The method described in the paragraph.   32. The method of claim 31, wherein the functional fragment is DNA encoding a CTGF module.   45. The method according to any one of claims 31 to 44, further comprising selecting the antibody-producing cell or the hybridoma by flow cytometry.

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