WO2019045086A1 - USE FOR SPECIFIC ANTIBODY FOR ACTIVE OR LATENT TGF-β1 - Google Patents

USE FOR SPECIFIC ANTIBODY FOR ACTIVE OR LATENT TGF-β1 Download PDF

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WO2019045086A1
WO2019045086A1 PCT/JP2018/032514 JP2018032514W WO2019045086A1 WO 2019045086 A1 WO2019045086 A1 WO 2019045086A1 JP 2018032514 W JP2018032514 W JP 2018032514W WO 2019045086 A1 WO2019045086 A1 WO 2019045086A1
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amino acid
acid sequence
seq
sequence represented
cdr
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隆之 水谷
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林化成株式会社
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the present invention relates to a monoclonal antibody that specifically binds to active transforming growth factor- ⁇ 1 (TGF- ⁇ 1) but not to latent TGF- ⁇ 1 or off-target protein, latency-associated peptide (LAP) and latent
  • TGF- ⁇ 1 active transforming growth factor- ⁇ 1
  • LAP latency-associated peptide
  • the present invention relates to a novel use of a monoclonal antibody that specifically binds to type TGF-.beta.1, but not to active TGF-.beta.1 and off-target proteins.
  • TGF- ⁇ 1 is a multifunctional cytokine belonging to the transforming growth factor spar family, which includes five isoforms (TGF- ⁇ 1 to ⁇ 5) and many other signaling proteins produced by the leucocyte cell lineage.
  • TGF-.beta Activated TGF-.beta., Together with other factors, form a serine / threonine kinase complex that binds to the TGF-.beta.
  • Receptor consisting of type 1 and type 2 receptor subunits.
  • type 2 receptor kinase phosphorylates and activates type 1 receptor kinase, thereby activating the signal transduction cascade and functioning in differentiation, migration, proliferation, activation of immune cells, etc.
  • Activation of downstream factors is triggered, including transcription of various target genes. Therefore, it is one of the cytokines that are highly studied in the fields of cancer, autoimmune diseases, infectious diseases and the like.
  • TGF- ⁇ isoforms are very similar (70-80% homology). They are all encoded as large precursor proteins, TGF- ⁇ 1 consists of 392 amino acids and TGF- ⁇ 2 and TGF- ⁇ 3 consist of 412 amino acids. TGF- ⁇ isoform has a signal peptide consisting of N-terminal 20-30 amino acids, a pro-region called LAP, and a C-terminal region consisting of 112-114 amino acids which are released from the pro-region by protease cleavage and become mature TGF- ⁇ molecules. Have. TGF- ⁇ homodimers interact with LAP to form a complex called Small Latent Complex (SLC).
  • SLC Small Latent Complex
  • LLC Large Latent Complex
  • ECM extracellular matrix
  • TGF- ⁇ 1 is known to be involved in the onset / progression of various diseases accompanied by fibrosis such as cancer and pulmonary fibrosis.
  • TGF- ⁇ 1 promotes epithelial-mesenchymal transition (EMT) of cancer cells, thereby imparting the cancer cells with motility and imparting advantageous properties to invasion and metastasis.
  • EMT epithelial-mesenchymal transition
  • TGF- ⁇ 1 not only supplies nutrients and oxygen to cancer cells but also provides a metastatic route by promoting angiogenesis.
  • mesenchymal precursor cells induce differentiation into fibroblasts and myofibroblasts that play an important role in cancer growth and metastasis.
  • TGF- ⁇ 1 present on the surface of cancer cell-derived exosomes suppresses the activation of CTL by antigen-presenting cells, and also acts on helper T cells, NK cells and macrophages in a suppressive manner. It has also been reported to destroy the protective immune mechanism against
  • Hematoxylin-eosin (HE) staining is a simple and general method for diagnosing cancer, but this technique alone can not be used to diagnose cancer malignancy and prognosis, so a treatment method is used.
  • diagnostic imaging such as CT is used in combination. Therefore, in order to evaluate the malignancy of cancer more easily, TGF- ⁇ 1 which is deeply involved in cancer invasion / metastasis and immunosuppression is detected by immunostaining using an antibody, Western blot, FACS, etc. Methods have been tried since before.
  • pancreatic cancer there is fibrosis that correlates with the function of the organ, and when fibrosis is brought about as a result of chronic inflammation, for example, liver cirrhosis in the liver and lung fibrosis in the lungs, such as liver dysfunction and respiratory function decline Get into a serious condition.
  • a tumor such as pancreatic cancer where the density of the tumor is very low, since the fiber component is formed in the stroma, it may be difficult to identify a tumor cell at the time of diagnosis, which makes it difficult to diagnose. Sometimes. Therefore, it is considered that the diagnostic ability of pancreatic cancer can be improved if fibrosis observed at the time of tumor formation is used as an index.
  • diagnosis of fibrosis can be conveniently performed by inducing differentiation of fibroblasts and myofibroblasts and detecting TGF- ⁇ 1 closely associated with fibrosis using an antibody.
  • an antibody with high specificity and high sensitivity to the extent that it can be used for diagnosis of cancer and fibrosis.
  • anti-TGF- ⁇ 1 monoclonal antibody "MAB240" product catalog Novusbio anti-TGF- ⁇ 1 monoclonal antibody "NB02-22114” product catalog SAB Biotech's anti-TGF- ⁇ 1 monoclonal antibody "44154” product catalog Santa Cruz, Inc., an anti-TGF- ⁇ 1 monoclonal antibody "sc-130348" product catalog LifeSpan BioSciences, anti-TGF- ⁇ 1 monoclonal antibody "LS-B9832" product catalog
  • the object of the present invention is that it is sufficiently sensitive and specific for either active TGF- ⁇ 1 or latent TGF- ⁇ 1 and LAP, and an off-target protein such as another TGF- ⁇ isoform It is also to develop a new method that can provide meaningful information in diagnosis of cancer and fibrosis using highly specific anti-TGF- ⁇ 1 antibody that does not cross-react either.
  • the present inventor specifically recognizes latent TGF- ⁇ 1 and LAP specifically with a novel monoclonal antibody (antibody (1)) that specifically recognizes active TGF- ⁇ 1, jointly developed by Bonak and AVNOVA.
  • antibody (1) a novel monoclonal antibody
  • antibody (2) a novel monoclonal antibody that specifically recognizes active TGF- ⁇ 1
  • excellent sensitivity stainability
  • cancer tissue normal gland tissue, muscle fibers, fibroblasts
  • antibody (1) in the stainability in cancer tissue and non-cancerous part
  • antibody (2) ie, the abundance ratio of active and latent TGF- ⁇ 1 differs depending on the tissue
  • samples from patients with cancer or fibrosis, or those suspected of: The following (a1) to (f1) complementarity determining regions (CDRs): (A1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 1, (B1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 2, (C1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 3, (D1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 4, (E1) CDR comprising the amino acid sequence represented by SEQ ID NO: 5, and (f1) CDR comprising the amino acid sequence represented by SEQ ID NO: 6
  • a test method for diagnosing cancer or fibrosis, or diagnosing cancer malignancy or progression of fibrosis which is characterized by specifically detecting ⁇ 1 or LAP.
  • the CDRs contained in the heavy chain variable region of the first monoclonal antibody are the above (a1), (b1) and (c1), and the CDRs contained in the light chain variable region are the above (d1), (e1) And (f1),
  • the CDRs contained in the heavy chain variable region of the second monoclonal antibody are the above (a2), (b2) and (c2), and the CDRs contained in the light chain variable region are the above (d2), (e2) and (f2) ), The method described in [1].
  • the CDR1, CDR2 and CDR3 contained in the heavy chain variable region of the first monoclonal antibody are the above (a1), (b1) and (c1) respectively, and the CDR1, CDR2 and CDR3 contained in the light chain variable region Are the above (d1), (e1) and (f1) respectively
  • the CDR1, CDR2 and CDR3 contained in the heavy chain variable region of the second monoclonal antibody are the above (a2), (b2) and (c2) respectively, and the CDR1, CDR2 and CDR3 contained in the light chain variable region are each mentioned above (D2), (e2) and (f2), The method described in [1].
  • the first monoclonal antibody is (X1) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13; and (Y1) a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14;
  • the second monoclonal antibody is (X2) The method according to [1], comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16 (Y2).
  • the first monoclonal antibody binds to active TGF- ⁇ 1 competitively with the first monoclonal antibody according to any of [1] to [4], and binds to latent TGF- ⁇ 1 Not an antibody
  • the second monoclonal antibody binds to latent TGF- ⁇ 1 or LAP in competition with the second monoclonal antibody according to any of [1] to [4], and does not bind to active TGF- ⁇ 1.
  • [6] The method according to any one of [1] to [5], wherein the sample is a biopsy tissue, a body fluid or an exosome.
  • the antibody of the present invention can detect active TGF- ⁇ 1 or latent TGF- ⁇ 1 / LAP with high sensitivity regardless of the type of tissue. In-situ analysis makes it possible to evaluate the degree of malignancy of a tumor (whether it is likely to metastasize or to invade surrounding tissues), and to evaluate whether it reflects the degree of response of therapeutic agents. In addition, the concentration of TGF- ⁇ 1 in blood is measured by ELISA or the like, and expression comparison analysis of active and latent forms enables judgment of malignancy. Furthermore, exosome analysis using FACS or MACS enables early cancer diagnosis and prognosis. Furthermore, according to the present invention, it is possible to diagnose fibrosis associated with a tumor and fibrosis in the whole body.
  • HE staining image immunostaining image using anti-active TGF- ⁇ 1 antibody as primary antibody
  • immunostaining image using anti-latent TGF- ⁇ 1 / LAP antibody as primary antibody negative control (no primary antibody ).
  • HE shows hematoxylin-eosin stained image
  • NC shows negative control (without primary antibody).
  • a gene or “DNA” is used to include not only double-stranded DNA but also single-stranded DNAs such as the sense strand and the antisense strand that constitute it. Also, the length is not particularly limited. Therefore, unless otherwise stated, a gene (DNA) in the present specification has a double-stranded DNA containing human genomic DNA, a single-stranded DNA (positive strand) containing cDNA, and a sequence complementary to the positive strand 1 Included are single stranded DNA (complementary strand), as well as any of these fragments.
  • TGF- ⁇ 1 gene refers to the cDNA sequence of the accession no. It means the human TGF- ⁇ 1 gene (DNA) registered as NM — 000660, its natural variant or polymorphic variant. Such variants or polymorphic variants include, for example, those registered in the SNP database available from NCBI.
  • TGF- ⁇ 1 protein or simply “TGF- ⁇ 1” have their amino acid sequences described in GenBank accession no. Human TGF- ⁇ 1 protein registered as NP_000651 (in the sequence, the signal peptide at position 1-29, LAP at position 30-278 and mature TGF- ⁇ 1 at position 279), or the natural mutation described above
  • NP_000651 in the sequence, the signal peptide at position 1-29, LAP at position 30-278 and mature TGF- ⁇ 1 at position 279
  • a protein encoded by a body or polymorphic variant DNA is meant.
  • latent TGF- ⁇ 1 refers to a mature TGF- ⁇ 1 homodimer and an inactive form of TGF- ⁇ 1 consisting of a homodimer of LAP bound thereto noncovalently, optionally further comprising LTBP.
  • Active TGF- ⁇ 1 means a homodimer of mature TGF- ⁇ 1 released from latent TGF- ⁇ 1.
  • antibody as used herein includes a part of the above-mentioned antibody having antigen binding ability, such as polyclonal antibody, monoclonal antibody, chimeric antibody, single chain antibody, or Fab fragment.
  • an epitope is a region of an antigen to which an antibody binds. In certain embodiments, it comprises any portion of an antigen that can specifically bind to an immunoglobulin.
  • An antigenic determinant comprises a chemically active surface group of molecules such as amino acids, sugar side chains, phosphoryl groups or sulfonyl groups, and in certain embodiments, specific three dimensional structural features and / or specific It may have charged features.
  • an antibody can be said to specifically bind an antigen if it preferentially recognizes a target antigen in a complex mixture of proteins and / or macromolecules.
  • the basic structure of the antibody molecule is common to all classes and is composed of a heavy chain having a molecular weight of 5 to 70,000 and a light chain having a molecular weight of 30,000 (I. Roitt, J. Brostoff, D. Male edition)).
  • the heavy chain usually consists of a polypeptide chain containing about 440 amino acids, and has a distinctive structure for each class, corresponding to IgG, IgM, IgA, IgD, and IgE, ⁇ , ⁇ , ⁇ , ⁇ It is called a chain.
  • IgG1, IgG2, IgG3 and IgG4 are present as IgG, and they are called ⁇ 1, ⁇ 2, ⁇ 3 and ⁇ 4, respectively.
  • the light chain is usually composed of a polypeptide chain containing about 220 amino acids, and two types, L-type and K-type, are known, which are called ⁇ and ⁇ chains, respectively.
  • the peptide composition of the basic structure of the antibody molecule is such that two heavy chains and two light chains, which are homologous to each other, are linked by disulfide bond (S-S bond) and non-covalent bond, and have a molecular weight of 15-190,000.
  • S-S bond disulfide bond
  • the two light chains can be paired with any heavy chain.
  • Each antibody molecule is always made up of two identical light chains and two identical heavy chains.
  • V regions variable regions
  • VH variable domain
  • VL variable domain
  • the amino acid sequence at the C-terminal side from this is almost constant for each class or subclass, and is called a constant region (C region) (each domain is represented as CH1, CH2, CH3 or CL, respectively).
  • the antigenic determination site of the antibody is constituted by VH and VL, and the specificity of binding depends on the amino acid sequence of this site.
  • biological activities such as complement and binding to various cells reflect differences in the structure of the C region of each class Ig.
  • CDRs complementarity determining regions
  • the part of the variable region other than the CDR is called a framework region (FR) and is relatively constant.
  • the framework regions adopt a beta sheet conformation, and the CDRs can form loops connecting beta sheet structures.
  • the CDRs in each chain retain their three dimensional structure by the framework regions and form an antigen binding site with the CDRs from the other chain.
  • the Kabat definition is based on sequence variability and the Chothia definition is based on the position of the structural loop region.
  • AbM definition is a compromise between the Kabat and Chothia approaches.
  • the CDRs of the light chain and heavy chain variable regions are demarcated according to the Kabat, Chothia or AbM algorithm (Martin et al. (1989) Proc. Natl. Acad. Sci. USA 86: 9268-9272; Martin et al. (1991) Methods Enzymol. 203: 121-153; Pedersen et al. (1992) Immunomethods 1: 126; and Rees et al. (1996) In Sternberg M. J. E. (ed.), Protein Structure Prediction. , Oxford University Press, Oxford, pp. 141-172).
  • the CDRs of the antibody of the present invention comprise the nucleotide sequences of the variable regions (VH and VL) of the heavy chain and light chain of the antibody, as well as public CDR determination software (http://www.abysis.org and http: // www It is defined as a CDR identified by analysis using .ncbi.nlm.nih.gov / igblast / igblast.cgi).
  • the CDRs of the heavy chain variable region are amino acids 26-32 (CDR1-H), 52-57 (CDR2-H), and the like in the amino acid sequence represented by SEQ ID NO: 13 99 to 109 (CDR 3-H), and the CDRs of the light chain variable region are amino acid Nos. 24 to 40 (CDR 1-L), 52 to 57 (CDR 2-L), and the amino acid sequences represented by SEQ ID NO: 14 99 to 102 (CDR 3-L).
  • the CDRs of the heavy chain variable region are amino acid Nos.
  • the confirmation of antibody binding can be carried out by any known assay method, such as direct and indirect sandwich assays, flow cytometry and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, ( CRC Press, Inc. 1987) pp. 147-158).
  • the binding of an anti-TGF- ⁇ 1 antibody to active TGF- ⁇ 1 or latent TGF- ⁇ 1 / LAP polypeptide can be measured, for example, according to the following method.
  • human TGF- ⁇ 1 / LAP polypeptide is adsorbed on a solid phase and blocked with a protein (skimmed milk, albumin, etc.) not involved in antigen-antibody reaction or enzyme reaction, and then anti-TGF- ⁇ 1 monoclonal
  • An antibody (test antibody) is brought into contact with a solid phase and incubated, and after removing an unreacted antibody by B / F separation, a labeled secondary antibody (anti-mouse IgG etc.) specifically reacting with the test antibody
  • a labeled secondary antibody specifically reacting with the test antibody
  • the solid phase for example, insoluble polysaccharides such as agarose, dextran and cellulose, plastics, polystyrene, polyacrylamide, synthetic resins such as silicon (tubes, microplates, etc.) or glass (beads, tubes etc.) etc.
  • radioactive isotopes for example, radioactive isotopes, enzymes, fluorescent substances, luminescent substances and the like are used.
  • radioactive isotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • enzyme one which is stable and has a large specific activity is preferable.
  • ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • light-emitting substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • a competitive assay such as a competitive ELISA can be used for identification.
  • the competition assay is carried out by causing free antigen or known antibody to coexist in the reaction system of the solid phase and the test antibody in the binding assay using the antigen-immobilized solid phase described above. For example, a test antibody solution of known concentration and a mixed solution obtained by adding antigens of various concentrations to the test antibody solution are brought into contact with an antigen-immobilized solid phase and incubated, and the labeled amount on the solid phase is measured respectively .
  • Scatchard analysis can be performed from the measured values at each free antigen concentration, and the slope of the graph can be calculated as a binding constant.
  • an antigen-immobilized solid phase is reacted with a labeled known antibody (the antibody of the present invention) and test antibodies of various concentrations to reduce the labeled amount on the solid phase in a concentration-dependent manner.
  • a test antibody it is possible to identify an antibody that binds to active or latent TGF- ⁇ 1 competitively with the antibody of the present invention.
  • the present invention provides a first monoclonal antibody that binds to active TGF- ⁇ 1 but does not bind to latent TGF- ⁇ 1 (hereinafter referred to as “the present monoclonal antibody A second monoclonal antibody (hereinafter referred to as “the antibody according to the present invention (hereinafter referred to as“ the antibody according to the invention (hereinafter referred to as “the antibody according to the invention (hereinafter referred to as“ antibody according to the invention 2) “test method for diagnosis of cancer or fibrosis, or diagnosis of cancer malignancy or progression of fibrosis (hereinafter referred to as“ test method according to the present invention, ”) Also referred to as The antibody (1) of the present invention and the antibody (2) of the present invention are collectively referred to as "the antibody of the present invention”.
  • the antibodies of the invention are further characterized by not recognizing any off-target proteins predicted from the epitopes recognized by the antibodies.
  • the antibody (1) of the present invention has (i) the following (a1) to (f1) complementarity determining regions (CDR): (A1) CDR comprising the amino acid sequence (SEQ ID NO: 1) represented by Gly Tyr Thr Phe Ser Asn Tyr (B1) CDR comprising the amino acid sequence (Sequence number 2) represented by Tyr Pro Gly Asn Ser Asp, (C1) a CDR comprising the amino acid sequence (SEQ ID NO: 3) represented by Tyr Ser Asn Tyr Glu Ala Gly Ala Met Asp Tyr; (D1) a CDR comprising the amino acid sequence (SEQ ID NO: 4) represented by Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Arg Lys Asn Tyr Leu Ala, (E1) CDR comprising the amino acid sequence (SEQ ID NO: 5) represented by Trp Ala Ser The Arg Glu Ser, and (f1) the amino acid sequence represented by Gln Gln Ser Tyr His Leu Pro Thr (SEQ ID NO: 6)
  • the antibody (1) of the present invention is (I) An antibody comprising a heavy chain variable region comprising the CDRs of (a1) to (c1) and a light chain variable region comprising the CDRs of (d1) to (f1), or (ii) the above (i) In the respective amino acid sequences of one or more (e.g., 1, 2, 3, 4, 5 or 6) CDRs selected from the above (a1) to (f1), including heavy and light chain variable regions of 1 or 2 amino acid residues are substituted and / or deleted and / or added and / or inserted), and an antibody that binds to active TGF- ⁇ 1 but not latent TGF- ⁇ 1.
  • an antibody comprising a heavy chain variable region comprising the CDRs of (a1) to (c1) and a light chain variable region comprising the CDRs of (d1) to (f1), or (ii) the above (i)
  • CDRs selected from the above (a1) to (f1) including heavy and light chain variable regions of 1 or 2 amino acid residues
  • the CDRs of (a1), (b1) and (c1) above are arranged in this order from the N-terminal side of the heavy chain. That is, the CDRs of (a1), (b1) and (c1) correspond to CDR1, CDR2 and CDR3 of the heavy chain, respectively.
  • the CDRs of (d1), (e1) and (f1) above are arranged in this order from the N-terminal side of the light chain. That is, the CDRs of (d1), (e1) and (f1) correspond to CDR1, CDR2 and CDR3 of the light chain, respectively.
  • the antibody (1) of the present invention is (I) an antibody comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14; or (ii) the weight of (i) above And 1 or more, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to several, in any one or both of SEQ ID NOs: 13 and 14 (Eg, 1, 2, 3, 4 or 5) amino acid residues are substituted and / or deleted and / or added and / or inserted, and bind to active TGF- ⁇ 1, Antibodies that do not bind to TGF-.beta.1.
  • the 50th residue is preferably Pro
  • the 50th residue is preferably Is Leu.
  • the antibody (1) of the present invention binds to active TGF- ⁇ 1 competitively with any of the above-mentioned anti-active TGF- ⁇ 1 antibodies and binds to latent TGF- ⁇ 1 Not an antibody.
  • Competitive binding of antibodies can be measured by the above-described competition assay.
  • the antibody (2) of the present invention has (i) the following (a2) to (f2) complementarity determining regions (CDR): (A2) CDR comprising the amino acid sequence (SEQ ID NO: 7) represented by Gly Tyr Thr Phe Thr Asp Tyr (B2) a CDR comprising the amino acid sequence (SEQ ID NO: 8) represented by Ile Pro Asn Ser Gly Gly, (C2) a CDR comprising the amino acid sequence (SEQ ID NO: 9) represented by Glu Ala Met Asp Tyr, (D2) a CDR comprising the amino acid sequence (SEQ ID NO: 10) represented by Arg Ala Ser Gln Ser Ile Arg Asn Lys Leu His, (E2) a CDR comprising the amino acid sequence (SEQ ID NO: 11) represented by Tyr Ala Ser Gln Ser Ile Ser; and (f 2) the amino acid sequence represented by Leu Gln Ser Asn Ser Trp Pro Leu Thr (SEQ ID NO 12) CDRs included Or (ii) one or more
  • the antibody (2) of the present invention is (I) An antibody comprising a heavy chain variable region containing the CDRs of (a2) to (c2) above and a light chain variable region containing the CDRs of (d2) to (f2) above, or (ii) the above (i) In the respective amino acid sequences of one or more (for example, 1, 2, 3, 4, 5 or 6) CDRs selected from the above (a2) to (f2), including the heavy chain and light chain variable regions of An antibody which binds to latent TGF- ⁇ 1 or LAP but does not bind to active TGF- ⁇ 1 in which 1 or 2 amino acid residues are substituted and / or deleted and / or added and / or inserted) is there.
  • the CDRs of (a2), (b2) and (c2) above are arranged in this order from the N-terminal side of the heavy chain. That is, the CDRs of (a2), (b2) and (c2) correspond to CDR1, CDR2 and CDR3 of the heavy chain, respectively.
  • the CDRs of (d2), (e2) and (f2) above are arranged in this order from the N-terminal side of the light chain. That is, the CDRs of (d2), (e2) and (f2) correspond to CDR1, CDR2 and CDR3 of the light chain, respectively.
  • the antibody (2) of the present invention is (I) an antibody comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16; or (ii) the weight of (i) above And 1 or more, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to several, in any one or both of SEQ ID NOs: 15 and 16. (Eg, 1, 2, 3, 4 or 5) amino acid residues are substituted and / or deleted and / or added and / or inserted, and bind to latent TGF- ⁇ 1 or LAP, , An antibody that does not bind to active TGF- ⁇ 1.
  • the antibody (2) of the present invention binds to latent TGF- ⁇ 1 or LAP in competition with any of the above-described anti-latent TGF- ⁇ 1 / LAP antibodies, and activates active TGF- It is an antibody which does not bind to ⁇ 1.
  • Competitive binding of antibodies can be measured by the above-described competition assay.
  • the isotype of the antibody is not particularly limited, but preferably it includes IgG, IgM or IgA, particularly preferably IgG.
  • the antibody of the present invention is not particularly limited as to the form of the molecule as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding an antigenic determinant (epitope), and a complete antibody molecule
  • CDR complementarity determining region
  • fragments such as Fab, Fab ', F (ab') 2 etc.
  • genetically engineered conjugate molecules such as scFv, scFv-Fc, minibody, diabody etc, or polyethylene glycol (PEG) etc.
  • PEG polyethylene glycol
  • the antibody of the present invention can be produced by an antibody production method known per se. Hereinafter, a method of preparing an immunogen for producing an antibody of the present invention, and a method of producing the antibody will be described.
  • the antigen used to prepare the antibody of the present invention may be whole latent or mature TGF- ⁇ 1 or whole LAP polypeptide, or a partial peptide thereof, or an antigenic determinant identical thereto. It is possible to use (synthetic) peptides having one or more kinds.
  • a partial peptide of mature TGF- ⁇ 1 or LAP polypeptide consisting of 6 to 15 amino acids is used as an immunogen.
  • evolutionarily conserved unique amino acid sequences within the regions of mature TGF- ⁇ 1 and LAP can be used as immunogens. Such evolutionarily conserved unique amino acid sequences can be predicted in silico using commercially available epitope prediction software.
  • the whole latent or mature TGF- ⁇ 1 or the whole LAP polypeptide, or a partial peptide thereof is prepared, for example, from (a) human tissues or cells using a known method or a method analogous thereto, (b) a peptide synthesizer, etc. (C) culturing a transformant containing a DNA encoding a latent or mature TGF- ⁇ 1 whole or a whole LAP polypeptide, or a partial peptide thereof; 2.) The whole latent or mature TGF- ⁇ 1 or the whole LAP polypeptide, or a nucleic acid encoding the partial peptide thereof, is prepared by biochemical synthesis using a cell-free transcription / translation system as a template.
  • the immunogen prepared as described above is used for warm-blooded animals, for example, intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, etc. Depending on the method of administration, it is administered to the site where antibody production is possible either alone or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance antibody production ability upon administration. The administration is usually performed once every 1 to 6 weeks, for a total of about 2 to 10 times.
  • a warm-blooded animal for example, a mouse, a rat, a rabbit, a goat, a monkey, a dog, a guinea pig, a hedge, a donkey, a chicken and the like are used, but a mouse, a rat and a rabbit are preferable.
  • the immunogen can be subjected to extracorporeal immunization.
  • animal cells used for extracorporeal immunization include lymphocytes isolated from human and the above-mentioned warm-blooded animals (preferably mice and rats), peripheral blood, spleen, lymph nodes and the like, preferably B lymphocytes and the like. .
  • spleen is removed from an animal of about 4 to 12 weeks of age and spleen cells are separated and an appropriate medium (eg, Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, Ham's F12 medium, etc.) After washing with the medium, the cells are suspended in a medium supplemented with fetal bovine serum (FCS; about 5 to 20%) containing an antigen, and cultured using a CO 2 incubator or the like for about 4 to 10 days.
  • FCS fetal bovine serum
  • the antigen concentration includes, for example, 0.05 to 5 ⁇ g, but is not limited thereto. It is preferable to prepare thymocyte culture supernatants of animals of the same strain (preferably about 1 to 2 weeks old) according to a conventional method and to add to the medium.
  • the immunization is carried out by adding a mildipeptide or the like) to the medium together with the antigen.
  • individuals or cell populations with elevated antibody titers are selected from warm-blooded animals (eg, mice, rats) or animal cells (eg, humans, mice, rats) immunized with an antigen. After harvesting the spleen or lymph node 2 to 5 days after the final immunization, or culturing it for 4 to 10 days after extracorporeal immunization, recover the cells, isolate the antibody-producing cells, and fuse this with myeloma cells. Production hybridomas can be prepared.
  • the antibody titer in serum can be measured, for example, by reacting a labeled antigen with an antiserum and then measuring the activity of the labeling agent bound to the antibody.
  • the myeloma cells are not particularly limited as long as they can produce hybridomas that secrete a large amount of antibodies, but those that do not themselves produce or secrete antibodies are preferable, and those with high cell fusion efficiency are more preferable. Also, in order to facilitate selection of hybridomas, it is preferable to use a HAT (hypoxanthine, aminopterin, thymidine) sensitive strain.
  • HAT hyperxanthine, aminopterin, thymidine
  • mouse myeloma cells include NS-1, P3U1, SP2 / 0, AP-1 and the like
  • rat myeloma cells include R210.
  • human myeloma cells include SKO-007, GM1500-6TG-2, LICR-LON-HMy2, UC729-6 and the like.
  • the fusion operation can be carried out according to known methods, for example, the method of Koehler and Milstein (Nature, vol. 256, p. 495 (1975)).
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and preferably PEG is used.
  • PEG polyethylene glycol
  • the molecular weight of PEG is not particularly limited, but low toxicity and relatively low viscosity PEG 1000 to PEG 6000 are preferable.
  • the PEG concentration is, for example, about 10 to 80%, preferably about 30 to 50%.
  • various buffers such as serum-free medium (eg RPMI 1640), complete medium containing about 5 to 20% serum, phosphate buffered saline (PBS), Tris buffer etc. may be used it can. If desired, DMSO (eg, about 10 to 20%) can also be added.
  • the pH of the fusion solution is, for example, about 4 to 10, preferably about 6 to 8.
  • the preferred ratio of the number of antibody-producing cells (splenocytes) to the number of myeloma cells is usually about 1: 1 to 20: 1, usually 20 to 40 ° C., preferably 30 to 37 ° C. and usually 1 to 10 minutes of incubation Cell fusion can be performed efficiently.
  • Antibody-producing cell lines can also be obtained by infecting antibody-producing cells with a virus capable of transforming lymphocytes to immortalize the cells.
  • a virus for example, Epstein-Barr (EB) virus and the like can be mentioned.
  • EB Epstein-Barr
  • a recombinant EB virus that retains the ability to immortalize B lymphocytes but lacks the ability of viral particles to replicate as an EB system without the possibility of viral contamination (eg, switches the transition from latent to lytic state) It is also preferred to use such as a deletion in the gene.
  • B lymphocytes can be easily transformed by using the culture supernatant.
  • the cells are cultured in, for example, serum and a medium supplemented with serum and penicillin / streptomycin (P / S) (eg RPMI 1640) or serum free medium to which cell growth factor has been added, and the culture supernatant is separated by filtration or centrifugation.
  • P / S serum and penicillin / streptomycin
  • the antibody-producing B lymphocytes are suspended at a suitable concentration (eg, about 10 7 cells / mL) and incubated usually at about 20 to 40 ° C., preferably 30 to 37 ° C. for about 0.5 to 2 hours.
  • Antibody-producing B cell lines can be obtained.
  • T lymphocytes When human antibody-producing cells are provided as mixed lymphocytes, most people have T lymphocytes that show toxicity to EB virus-infected cells, so to increase transformation frequency, for example, T lymphocytes are preferably removed in advance by forming E rosettes with sheep red blood cells and the like.
  • lymphocytes specific to a target antigen can be selected by mixing soluble erythrocyte-bound sheep red blood cells with antibody-producing B lymphocytes and separating rosettes using a density gradient such as Percoll.
  • antigen-specific B lymphocytes can be sorted from this mixture by collecting a rosette non-forming layer using a density gradient such as Percoll.
  • Human antibody-secreting cells that have acquired infinite proliferation ability by transformation can be back-fused with mouse or human myeloma cells in order to stably sustain antibody secreting ability.
  • the same myeloma cells as those described above can be used.
  • Hybridomamas Screening and breeding of hybridomas are usually performed by adding HAT (hypoxanthine, aminopterin, thymidine) and using a medium for animal cells (eg RPMI 1640) containing 5-20% FCS or a serum-free medium supplemented with cell growth factors. It will be.
  • the concentrations of hypoxanthine, aminopterin and thymidine include, for example, about 0.1 mM, about 0.4 ⁇ M and about 0.016 mM, respectively.
  • ouabain resistance can be used. Since human cell lines are more sensitive to ouabain than mouse cell lines, unfused human cells can be excluded by adding to the medium at about 10 -7 to 10 -3 M.
  • feeder cells it is preferable to use feeder cells and certain cell culture supernatants for selection of hybridomas.
  • feeder cells allogeneic cell types with a limited survival time to help the emergence of hybridomas and die themselves, irradiation with cells that can produce large amounts of growth factors useful for emergence of hybridomas, etc. Those having reduced proliferation ability are used.
  • mouse feeder cells include splenocytes, macrophages, blood, thymocytes and the like
  • human feeder cells include peripheral blood mononuclear cells and the like.
  • the cell culture supernatant include primary culture supernatants of the various cells described above and culture supernatants of various established cell lines.
  • hybridomas can also be selected by fluorescently labeling the antigen and reacting it with the fused cells, and then separating out cells that bind to the antigen using a fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • Various methods can be used to clone a hybridoma producing a monoclonal antibody against a target antigen.
  • aminopterin inhibits many cellular functions, it is preferable to remove it from the medium as soon as possible. In the case of mice and rats, most myeloma cells die within 10-14 days, and aminopterin can be removed two weeks after fusion. However, human hybridomas are usually maintained in an aminopterin-containing medium for about 4 to 6 weeks after fusion. It is desirable to remove hypoxanthine and thymidine one week or more after aminopterin removal. That is, in the case of mouse cells, for example, addition or replacement of hypoxanthine and thymidine (HT) -added complete medium (eg, RPMI 1640 supplemented with 10% FCS) is performed 7 to 10 days after fusion. Visually visible clones appear about 8 to 14 days after fusion. When the diameter of the clone is about 1 mm, the amount of antibody in the culture supernatant can be measured.
  • hypoxanthine and thymidine (HT) -added complete medium eg,
  • the antibody amount can be measured, for example, by using a hybridoma culture supernatant on a solid phase (eg, microplate) on which a target antigen or a derivative thereof or a partial peptide thereof (including a partial amino acid sequence used as an antigenic determinant) is adsorbed directly or with a carrier.
  • a hybridoma culture supernatant on a solid phase eg, microplate
  • a target antigen or a derivative thereof or a partial peptide thereof including a partial amino acid sequence used as an antigenic determinant
  • radioactive substances eg 125 I, 131 I, 3 H, 14 C
  • enzymes eg ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase
  • fluorescence Anti-immunoglobulin (IgG) antibodies eg, from which the original antibody-producing cells are labeled
  • substances eg, fluorescamine, fluorescein isothiocyanate
  • luminescent substances eg, luminol, luminol derivatives, luciferin, lucigenin
  • Antibodies against IgG from the same kind of animals are used
  • Method for detecting protein against target antigen (antigenic determinant) bound to solid phase by adding protein A, adding hybridoma culture supernatant to the solid phase adsorbed with anti-IgG antibody or protein A, and labeling as described above It can be carried out by a method of detecting an antibody against a target antigen (antigenic determinant) bound to a solid phase by adding an agent-labeled target antigen or a derivative thereof or a partial peptide thereof.
  • cloning using soft agar and cloning using FACS are also possible.
  • the cloning by the limiting dilution method can be performed, for example, by the following procedure, but is not limited thereto.
  • the amount of antibody is measured as described above to select positive wells.
  • Appropriate feeder cells are selected and added to the 96 well plate.
  • Aspirate cells from antibody-positive wells suspend in complete medium (eg 10% FCS and P / S supplemented RMPI 1640) to a density of 30 cells / mL, and add 0.1 mL to the well plate to which feeder cells have been added.
  • Add 3 cells / well dilute the remaining cell suspension to 10 cells / mL, and in the same way to another well (1 cell / well), further dilute the remaining cell suspension to 3 cells / mL Then in another well (0.3 cells / well).
  • the cells are cultured for about 2 to 3 weeks until visible clones appear, the amount of antibody is measured, and positive wells are selected and cloned again. Plates of 10 cells / well are also prepared because cloning is relatively difficult in the case of human cells. Although monoclonal antibody-producing hybridomas can usually be obtained by two subclonings, it is desirable to carry out periodical reclonings for several more months to confirm the stability.
  • a hybridoma that produces a monoclonal antibody that reacts with active TGF- ⁇ 1 and / or its partial peptide but does not react with latent TGF- ⁇ 1 is referred to as anti-active TGF- of the present invention. It can be selected as a hybridoma that produces ⁇ 1 antibody.
  • a hybridoma producing a monoclonal antibody which reacts with latent TGF- ⁇ 1 and / or a partial peptide of LAP but does not react with active TGF- ⁇ 1 is used as an anti-tumor agent of the present invention. It can be selected as a hybridoma that produces latent TGF- ⁇ 1 / LAP antibody.
  • the antibody of the present invention does not cross react with any off-target proteins, including TGF- ⁇ 2, TGF- ⁇ 3, etc. as predicted from the amino acid sequence of the epitope that it recognizes. Therefore, hybridomas producing monoclonal antibodies that recognize only either active TGF- ⁇ 1 or latent TGF- ⁇ 1 / LAP obtained as described above are subjected to negative selection, and they cross react with off-target proteins. You can confirm that you do not.
  • the hybridoma thus obtained can be cultured in vitro or in vivo.
  • the monoclonal antibody-producing hybridoma obtained as described above may be used as a well while maintaining the cell density at, for example, about 10 5 to 10 6 cells / mL and gradually reducing the FCS concentration.
  • mice that have been injected with mineral oil into the peritoneal cavity to induce plasmacytoma (MOPC) (10 mice that are histocompatibility with the parent strain of the hybridoma) may be 10 6 days later.
  • MOPC plasmacytoma
  • (D) Purification of monoclonal antibody Separation and purification of monoclonal antibody can be carried out by a method known per se, for example, separation and purification of immunoglobulin [eg salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchange
  • the antibody alone is collected by adsorption / desorption method by the body (eg DEAE, QEAE), ultracentrifugation, gel filtration, antigen binding solid phase or active adsorbent such as protein A or protein G, and the binding is dissociated to obtain antibody.
  • a monoclonal antibody can be produced by culturing a hybridoma in or on a warm-blooded animal and collecting the antibody from its body fluid or culture.
  • Examples of the antibody (1) of the present invention include mouse anti-active TGF- ⁇ 1 antibody clones 2H4 and 4D10 described in the Examples below.
  • Examples of the antibody (2) of the present invention a mouse anti-latent TGF- ⁇ 1 antibody clone 2F10 described in the following Examples can be mentioned.
  • the 2H4 and 4D10 antibodies consist of the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 14 (however, 11th and 50th in the case of 2H4 antibody)
  • the th residue is Leu and Pro, respectively, and in the case of the 4D10 antibody, it has been revealed that the 11th and 50th residues are Arg and Leu, respectively, and a light chain variable region.
  • the 2F10 antibody was also found to have a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16.
  • the cDNA encoding the heavy chain and light chain of the thus-obtained anti-active TGF- ⁇ 1 antibody or anti-latent TGF- ⁇ 1 / LAP antibody is used as the antibody. It can be isolated from a cDNA library of producing hybridomas and cloned into an appropriate expression vector functional in a desired host cell according to a conventional method. The heavy and light chain expression vectors thus obtained are then introduced into host cells.
  • Useful host cells include animal cells, such as mouse myeloma cells as described above, Chinese hamster ovary (CHO) cells, COS-7 cells from monkeys, Vero cells, GHS cells from rats, etc.
  • any method applicable to animal cells may be used, but preferably, electroporation, a method using a cationic lipid, and the like can be mentioned.
  • the culture supernatant can be recovered and the antibody protein can be isolated by conventional purification of the antibody protein.
  • transgenic animals are produced by a conventional method using germline cells of animals in which transgenic techniques such as cattle, goats and chickens have been established as host cells, and know-how on mass breeding has been accumulated as livestock (poultry). By doing this, the antibody of the present invention can also be obtained easily and in large amounts from the milk or egg of the animal obtained.
  • plant cells that have been established as transgenic plants such as maize, rice, wheat, soybean, and tobacco, and are grown in large quantities as major crops, are used as host cells for microinjection and electroporation into protoplasts, and intact cells. It is also possible to produce a transgenic plant using the particle gun method or Ti vector method of the above, and to obtain the antibody of the present invention in large amounts from the resulting seeds and leaves.
  • the test method of the present invention binds to active TGF- ⁇ 1 with a sample from a cancer or fibrotic patient or a person suspected of it, but latent TGF- ⁇ 1
  • a first monoclonal antibody (antibody (1) of the present invention) that does not bind to the antibody, and / or a second monoclonal antibody that binds to latent TGF- ⁇ 1 or LAP but not to active TGF- ⁇ 1 ( It is characterized in that it is contacted with the antibody (2) of the present invention to specifically detect active TGF- ⁇ 1 and / or latent TGF- ⁇ 1 or LAP in the sample.
  • the “cancer” to be subjected to the test method of the present invention is not particularly limited, and may be, for example, an epithelial cell-derived cancer, but may be non-epithelial sarcoma or hematologic cancer. More specifically, for example, cancer of the head and neck (eg, maxillary cancer, pharyngeal cancer, laryngeal cancer, tongue cancer, thyroid cancer), breast cancer (eg, breast cancer, lung cancer (non-small) Cell lung cancer, small cell lung cancer), digestive tract cancer (eg esophagus cancer, stomach cancer, duodenal cancer, colon cancer (colon cancer, rectum cancer), liver cancer (hepatocellular carcinoma, bile duct) Cell cancer), gallbladder cancer, bile duct cancer, pancreatic cancer, anal cancer), urinary cancer (eg, renal cancer, ureteral cancer, bladder cancer, prostate cancer, penile cancer, Testicular (testicular cancer), cancer of the genital organs (eg, uterine
  • the “fibrosis” to be subjected to the test method of the present invention is not particularly limited as long as it is a disease involving tissue fibrosis, and for example, pulmonary fibrosis, hepatic fibrosis, cirrhosis, nonalcoholic steatohepatitis (NASH), Chronic hepatitis C, ulcerative colitis, Crohn's disease and the like can be mentioned, but it is not limited thereto.
  • the test method of the present invention can also be used for diagnosis of tumor stromal fibrosis in the above-mentioned various cancers.
  • cancer patient and “fibrosis patient” are confirmed to be suffering from any of the above-mentioned cancers or fibrosis by a definitive diagnostic method currently used clinically.
  • the “suspected person” of cancer or fibrosis does not have a definitive diagnosis, but suffers from any of the above cancers or fibrosis by any test, medical examination, etc. It means a person who is predicted to have a risk of developing it in the future.
  • the sample is not particularly limited as long as it is a sample that can be diagnosed as having malignancy or progression of fibrosis, and examples thereof include biopsy tissue, body fluid, exosome and the like.
  • the body fluid include blood (eg, whole blood, serum, plasma), urine, ascites fluid, vaginal lavage fluid, saliva, cerebrospinal fluid and the like.
  • it is serum, plasma or the like.
  • exosome the exosome contained in said various bodily fluid is mentioned.
  • they are exosomes separated from serum or plasma.
  • the exosome can be separated and purified by ultracentrifugation, equilibrium density gradient centrifugation, immunological capture, size exclusion, phospholipid affinity, polymer precipitation, etc. which are known per se.
  • TGF- ⁇ 1 promotes epithelial-mesenchymal transition (EMT) of cancer cells, thereby imparting the cancer cells with motility and imparting advantageous properties to invasion and metastasis.
  • EMT epithelial-mesenchymal transition
  • TGF- ⁇ 1 promotes angiogenesis to contribute to cancer cell survival and to provide a metastatic route.
  • it induces differentiation of mesenchymal precursor cells into fibroblasts and myofibroblasts in tumor stroma, and promotes proliferation and metastasis of cancer.
  • TGF- ⁇ 1 on the surface of cancer cell-derived exosomes activates CTL, suppresses the functions of helper T cells, NK cells, and macrophages, and invalidate cancer protective immunity.
  • latent TGF- ⁇ 1 does not express these functions because the various TGF- ⁇ 1 functions mentioned above are triggered by stimulating active TGF- ⁇ 1 with a TGF- ⁇ receptor. Therefore, if each expression level of active and latent TGF- ⁇ 1 can be accurately measured in the cancer tissue and its surrounding tissue, the malignancy (eg, easy metastatic, easily invasive) or fibrosis of the cancer It is possible to distinguish the degree of progression of
  • conventionally known anti-TGF- ⁇ 1 antibodies have very poor detection sensitivity depending on the sample (for example, the type of tissue) and can detect TGF- ⁇ 1 in a sample that should be positive in nature Or there is a problem with specificity such as being unable to distinguish and detect active TGF- ⁇ 1 and latent TGF- ⁇ 1 or cross-reacting with off-target proteins, etc.
  • the antibody of the present invention is produced using the evolutionarily conserved unique partial amino acid sequence as an immunogen for each of mature TGF- ⁇ 1 and LAP, activated TGF- ⁇ 1 or Only one of latent TGF- ⁇ 1 / LAP can be specifically recognized, and therefore, the expression level of each of activated TGF- ⁇ 1 or latent TGF- ⁇ 1 / LAP in a sample is accurately measured. The risk of false positives is significantly improved, as it is possible and does not cross react with off-target proteins.
  • Detection of active TGF- ⁇ 1 and / or latent TGF- ⁇ 1 or LAP in a sample using the antibody (1) and / or (2) of the present invention is a suitable antigen depending on the type of sample.
  • An antibody response assay can be selected as appropriate. For example, in the case where the sample is a biopsy tissue collected from a subject, well-known conventional immunohistological staining can be used.
  • radioactive isotopes for example, radioactive isotopes, enzymes, fluorescent substances, luminescent substances and the like are used.
  • a radioactive isotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • the above-mentioned enzyme one which is stable and has a large specific activity is preferable.
  • ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiocyanate (FITC), phycoerythrin (PE) or the like is used.
  • light-emitting substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • the antibody of the present invention may be directly labeled with a labeling substance, or may be labeled indirectly.
  • the antibody of the present invention is a non-labeled antibody (primary antibody), and a labeled secondary antibody such as an antiserum or an anti-Ig antibody to an animal in which the antibody of the present invention is produced - ⁇ 1 can be detected.
  • a biotinylated secondary antibody can be used to form a complex of TGF-.beta.1-antibody-secondary antibody of the present invention, which can be visualized using labeled streptavidin.
  • a biopsy tissue sample is fixed / permeabilized with glutaraldehyde, paraformaldehyde or the like, washed with a buffer such as PBS, blocked with BSA or the like, and then incubated with the antibody of the present invention. After washing with a buffer solution such as PBS to remove unreacted antibody, the tissue reacted with the present invention is visualized with a labeled secondary antibody, and visually observed under a microscope, or a confocal laser scanning microscope or Quantitative analysis using an automated live cell image analyzer such as IN Cell Analyzer (Amarsham / GE).
  • a buffer solution such as PBS
  • cancer tissues and benign tissues are both stained, but typically, with the antibody (1) of the present invention, a site of strong inflammation even in cancer tissue or benign tissue And in areas where fibrosis is prominent, staining is strong in benign tissues and is relatively weak in benign tissues, whereas in the case of the antibody (2) of the present invention, the staining property of cancerous tissue is the antibody (1) of the present invention It is weaker than the above, and in benign tissues, it shows the staining property equal to or more than that of the antibody (1) of the present invention.
  • active TGF- ⁇ 1 in cancer tissues, active TGF- ⁇ 1 is highly expressed, while in benign tissues, expression of latent TGF- ⁇ 1 is considered to be relatively high. If the expression of active TGF- ⁇ 1 is more pronounced in cancer tissues and their surrounding tissues (in particular, sites of strong inflammation and sites of marked fibrosis) compared to the expression of latent TGF- ⁇ 1,
  • the examiner's cancer can be predicted to be of high grade (eg, prone to distant metastasis, prone to invade surrounding tissues). Alternatively, if differentiation into fibroblasts or myofibroblasts is promoted in the tumor stroma and high expression of latent and / or active TGF- ⁇ 1 is observed at the relevant site, the subject is a tumor It can be predicted that associated fibrosis is developing.
  • the subject is a fibrotic patient or a person suspected of having the same, the subject is also fibrotic if high expression of latent and / or active TGF- ⁇ 1 is observed in the sample tissue. It can be predicted that there is a high risk of developing or a future onset of fibrosis, or that fibrosis is progressing.
  • the comparison of staining property may be made between the lesioned part and non-lesioned part of the sample tissue, or may be made between samples collected in time series from the same subject.
  • the presence or absence of onset or the onset risk can be determined by comparison with a corresponding sample derived from a healthy subject.
  • TGF- ⁇ 1 secreted from cancer cells leaks into blood and other body fluids, and on the membrane surface of cancer cell-derived exosomes. Although it is embedded and transported into body fluid, the expression level of active TGF- ⁇ 1 in blood and exosomes is considered to be correlated with the malignancy of cancer, so that active TGF- ⁇ 1 in these samples is
  • the expression level is measured using the antibody (1) of the present invention, and compared with a control (eg, a sample derived from a non-metastatic cancer patient), or the expression level of latent TGF- ⁇ 1 in the same sample is
  • the malignancy of a cancer in a subject can be predicted by measuring using the antibody (2) of the invention and comparing the abundance ratio of active TGF- ⁇ 1 and latent TGF- ⁇ 1.
  • the degree of progression of fibrosis can also be predicted.
  • Various immunoassays such as ELISA and fluorescence immunoassay known per se can be used to measure blood TGF- ⁇ 1.
  • proteins can be extracted from serum or plasma by a conventional method, and TGF- ⁇ 1 levels can be measured by Western blot analysis.
  • an antibody of the present invention is immobilized on magnetic beads, and an exosome presenting TGF- ⁇ 1 on the surface is separated from the sample by applying a magnetic field (ie, magnetic It can be made to activate cell separation (MACS).
  • a magnetic field ie, magnetic It can be made to activate cell separation (MACS).
  • the antibody of the present invention is directly or indirectly labeled with any suitable fluorescent molecule as described above, and a TGF-activated cell sorter (FACS) is used to display TGF- ⁇ 1 on the surface
  • FACS TGF-activated cell sorter
  • the level of latent TGF- ⁇ 1 is examined using the antibody (2) of the present invention, or activated TGF in combination with the antibody (1) of the present invention
  • the level of ⁇ 1 is examined using the antibody (2) of the present invention, or activated TGF in combination with the antibody (1) of the present invention
  • kits of the Present Invention also provides a diagnosis of cancer or fibrosis, or cancer malignancy or fibrosis comprising the antibody (1) of the present invention and the antibody (2) of the present invention Provide a diagnostic kit for progression.
  • the kit may further contain other known components suitable for carrying out the above-described test method of the present invention.
  • Example 1 Preparation of Antibody of the Present Invention
  • Immunogen An evolutionarily conserved unique sequence (10 amino acids) located in each of LAP and mature TGF- ⁇ 1 region of latent TGF- ⁇ 1 polypeptide It was extracted using commercially available epitope prediction software "Epitope Hunter". Peptides consisting of the extracted sequences were synthesized using an automatic peptide synthesizer, and they were linked to a carrier to make an immunogen.
  • HRP horseradish peroxidase
  • TGF-.beta.1 Cells or tissue lysates overexpressing TGF-.beta.1 were subjected to SDS-polyacrylamide electrophoresis, reacted with 5-fold diluted hybridoma culture supernatant, and immunoreactivity was visualized using HRP-labeled goat anti-mouse IgG. Ten out of eleven clones were positive for both cell and tissue lysates. In addition, fluorescent immunoassay was performed on HepG2 cells. Six out of ten clones showed a strong positive signal. Further detailed analysis was performed using 2 clones (2H4 and 4D10) of these. Similarly, anti-latent TGF- ⁇ 1 / LAP monoclonal antibody clone (2F10) was obtained from mice immunized with a partial peptide of LAP.
  • Example 2 Immunostaining of colorectal cancer tissue specimens using anti-active TGF- ⁇ 1 antibody and anti-latent TGF- ⁇ 1 / LAP antibody (1) Reagents used, kit VECTASTAIN ABC KIT Mouse IgG (PK-4002) ⁇ ImmPACT DAB Peroxidase Substrate Kit (SK-4105) ⁇ Various primary antibodies (antibodies prepared in Example 1 and commercially available antibodies manufactured by Santa Cruz, Matt Bio, LS Bio, PeproTech, Signal and R & D) ⁇ Hydrogen peroxide water, methanol ⁇ PBS Sodium citrate, trisodium citrate dihydrate Xylol, Ethanol Mayer's Hematoxylin
  • TGF- ⁇ 1 can not be detected in cancer tissues when using an antibody other than PeproTech's antibody.
  • the anti-latent TGF- ⁇ 1 / LAP antibody of the present invention obtained in Example 1 was used as a primary antibody, TGF- ⁇ 1 could be detected even in cancer tissues.
  • TGF-.beta.1 is also involved in fibrosis other than cancer, and fibroblasts and myofibroblasts, which are closely related to fibrosis, were also detected in TGF-.beta.1.
  • the present invention it is possible to determine the grade of a tumor (whether it is likely to be distant metastasis or to invade surrounding tissues) and to evaluate the efficacy of a therapeutic agent. Also, by measuring TGF- ⁇ 1 levels in the blood or exosome surface and comparing the expression of active and latent forms, judgment of malignancy of cancer, early cancer diagnosis, prognosis prediction, etc. become possible. In addition, diagnosis of fibrosis associated with tumor and systemic fibrosis is also possible. Therefore, the test method and test kit of the present invention are extremely useful clinically.

Abstract

The present invention provides an examination method for diagnosing cancer or fibrosis or diagnosing cancer malignancy or fibrosis progress, said examination method being characterized in that a sample from a person who has or may have cancer or fibrosis is made to come into contact with a first monoclonal antibody, which binds to active TGF-β1 but does not bind to latent TGF-β1 and includes (a1) a complementarity determining region (CDR) including the amino acid sequence represented by sequence number 1, (b1) a CDR including the amino acid sequence represented by sequence number 2, (c1) a CDR including the amino acid sequence represented by sequence number 3, (d1) a CDR including the amino acid sequence represented by sequence number 4, (e1) a CDR including the amino acid sequence represented by sequence number 5, and (f1) a CDR including the amino acid sequence represented by sequence number 6, and/or a second monoclonal antibody, which binds to latent TGF-β1 and LAP but does not bind to active TGF-β1 and includes (a2) a CDR including the amino acid sequence represented by sequence number 7, (b2) a CDR including the amino acid sequence represented by sequence number 8, (c2) a CDR including the amino acid sequence represented by sequence number 9, (d2) a CDR including the amino acid sequence represented by sequence number 10, (e2) a CDR including the amino acid sequence represented by sequence number 11, and (f2) a CDR including the amino acid sequence represented by sequence number 12, and the active TGF-β1 and/or latent TGF-β1 or LAP in the sample is specifically detected.

Description

活性型もしくは潜在型ＴＧＦ−β１特異的抗体の用途Use of Active or Latent TGF-β 1 Specific Antibody
　本発明は、活性型トランスフォーミング増殖因子−β１（ＴＧＦ−β１）に特異的に結合するが潜在型ＴＧＦ−β１やオフターゲットタンパク質には結合しないモノクローナル抗体と、ｌａｔｅｎｃｙ−ａｓｓｏｃｉａｔｅｄ　ｐｅｐｔｉｄｅ（ＬＡＰ）及び潜在型ＴＧＦ−β１に特異的に結合するが、活性型ＴＧＦ−β１及びオフターゲットタンパク質には結合しないモノクローナル抗体の新規用途に関する。 The present invention relates to a monoclonal antibody that specifically binds to active transforming growth factor-β1 (TGF-β1) but not to latent TGF-β1 or off-target protein, latency-associated peptide (LAP) and latent The present invention relates to a novel use of a monoclonal antibody that specifically binds to type TGF-.beta.1, but not to active TGF-.beta.1 and off-target proteins.
　ＴＧＦ−β１は、５種のアイソフォーム（ＴＧＦ−β１~β５）及び白血球細胞系譜により産生される他の多くのシグナル伝達タンパク質を含むトランスフォーミング増殖因子スパーファミリーに属する多機能性のサイトカインである。活性化したＴＧＦ−βは、他の因子とともに、１型及び２型の受容体サブユニットからなるＴＧＦ−β受容体に結合するセリン／スレオニンキナーゼ複合体を形成する。ＴＧＦ−βが結合すると、２型受容体キナーゼが１型受容体キナーゼをリン酸化して活性化し、それによってシグナル伝達カスケードが活性化され、分化、遊走、増殖、免疫細胞の活性化などに機能する種々の標的遺伝子の転写を含む、下流因子の活性化が引き起こされる。そのため、がん、自己免疫疾患、感染症などの分野で高度に研究されているサイトカインの１つである。 TGF-β1 is a multifunctional cytokine belonging to the transforming growth factor spar family, which includes five isoforms (TGF-β1 to β5) and many other signaling proteins produced by the leucocyte cell lineage. Activated TGF-.beta., Together with other factors, form a serine / threonine kinase complex that binds to the TGF-.beta. Receptor consisting of type 1 and type 2 receptor subunits. When TGF-β is bound, type 2 receptor kinase phosphorylates and activates type 1 receptor kinase, thereby activating the signal transduction cascade and functioning in differentiation, migration, proliferation, activation of immune cells, etc. Activation of downstream factors is triggered, including transcription of various target genes. Therefore, it is one of the cytokines that are highly studied in the fields of cancer, autoimmune diseases, infectious diseases and the like.
　ＴＧＦ−βアイソフォームの構造は非常に類似している（７０~８０％の相同性）。それらはいずれも大きな前駆体タンパク質としてコードされており、ＴＧＦ−β１は３９２アミノ酸、ＴＧＦ−β２及びＴＧＦ−β３は４１２アミノ酸からなる。ＴＧＦ−βアイソフォームは、Ｎ末端の２０~３０アミノ酸からなるシグナルペプチド、ＬＡＰと呼ばれるプロ領域、並びにプロテアーゼ切断によりプロ領域から遊離し成熟ＴＧＦ−β分子となる１１２~１１４アミノ酸からなるＣ末端領域を有する。ＴＧＦ−βホモダイマーはＬＡＰと相互作用してＳｍａｌｌ　Ｌａｔｅｎｔ　Ｃｏｍｐｌｅｘ（ＳＬＣ）と呼ばれる複合体を形成する。この複合体は潜在型ＴＧＦ−β結合タンパク質（ＬＴＢＰ）と呼ばれる別のタンパク質が結合するまで細胞内にとどまり、Ｌａｒｇｅ　Ｌａｔｅｎｔ　Ｃｏｍｐｌｅｘ（ＬＬＣ）と呼ばれるより大きな複合体を形成し、細胞外マトリクス（ＥＣＭ）に分泌する。たいていの場合、ＬＬＣが分泌する前に、ＴＧＦ−β前駆体はプロペプチドから切断されるが、非共有結合によりプロペプチドと結合したままである。分泌後、ＬＬＣは、ＬＴＢＰとＬＡＰの両方を含む不活性な複合体としてＥＣＭにとどまる。ＴＧＦ−βとＬＴＢＰとの結合はジスルフィド結合であり、それにより受容体への結合を阻止し不活性な状態にとどめる。 The structures of TGF-β isoforms are very similar (70-80% homology). They are all encoded as large precursor proteins, TGF-β1 consists of 392 amino acids and TGF-β2 and TGF-β3 consist of 412 amino acids. TGF-β isoform has a signal peptide consisting of N-terminal 20-30 amino acids, a pro-region called LAP, and a C-terminal region consisting of 112-114 amino acids which are released from the pro-region by protease cleavage and become mature TGF-β molecules. Have. TGF-β homodimers interact with LAP to form a complex called Small Latent Complex (SLC). This complex remains in the cell until another protein called latent TGF-beta binding protein (LTBP) binds, forming a larger complex called Large Latent Complex (LLC), extracellular matrix (ECM) Secret to In most cases, before LLC is secreted, the TGF-β precursor is cleaved from the propeptide but remains noncovalently bound to the propeptide. After secretion, LLC remains in the ECM as an inactive complex containing both LTBP and LAP. The bond between TGF-β and LTBP is a disulfide bond, thereby blocking binding to the receptor and leaving it inactive.
　ところで、ＴＧＦ−β１は、がんや肺線維症などの線維化を伴う種々の疾患の発症／進展に関与していることが知られている。例えば、ＴＧＦ−β１はがん細胞の上皮間葉転換（ＥＭＴ）を促進することで、がん細胞に運動能を付与し、浸潤・転移に有利な特性を持たせる。また、ＴＧＦ−β１は、血管新生を促進することで、がん細胞に栄養や酸素を供給するだけでなく、転移ルートを提供する。さらに、腫瘍間質において、間葉系前駆細胞から、がんの増殖・転移に重要な役割を果たす線維芽細胞、筋線維芽細胞への分化を誘導する。また、がん細胞由来エクソソームの表面に存在するＴＧＦ−β１が、抗原提示細胞によるＣＴＬの活性化を抑制し、ヘルパーＴ細胞やＮＫ細胞、マクロファージに対しても抑制的に作用して、がんに対する防御免疫機構を破壊することも報告されている。 By the way, TGF-β1 is known to be involved in the onset / progression of various diseases accompanied by fibrosis such as cancer and pulmonary fibrosis. For example, TGF-β1 promotes epithelial-mesenchymal transition (EMT) of cancer cells, thereby imparting the cancer cells with motility and imparting advantageous properties to invasion and metastasis. In addition, TGF-β1 not only supplies nutrients and oxygen to cancer cells but also provides a metastatic route by promoting angiogenesis. Furthermore, in tumor stroma, mesenchymal precursor cells induce differentiation into fibroblasts and myofibroblasts that play an important role in cancer growth and metastasis. In addition, TGF-β1 present on the surface of cancer cell-derived exosomes suppresses the activation of CTL by antigen-presenting cells, and also acts on helper T cells, NK cells and macrophages in a suppressive manner. It has also been reported to destroy the protective immune mechanism against
　がんの診断法としては、ヘマトキシリン−エオシン（ＨＥ）染色による診断が簡便で一般的であるが、この技術だけではがんの悪性度や予後予測の診断までは不可能なため、治療方法を選択する際にはＣＴなどの画像診断が併用されている。そこで、より簡便にがんの悪性度を評価するために、がんの浸潤・転移や免疫抑制に深く関わっているＴＧＦ−β１を、抗体を用いて免疫染色、ウエスタンブロット、ＦＡＣＳなどにより検出する方法が、以前より試みられてはいる。
　一方、臓器の機能と相関するものとして線維化があり、慢性炎症の結果線維化が招来すると、例えば肝臓であれば肝硬変、肺であれば肺線維症になり、肝機能不全や呼吸機能低下といった重篤な状態に陥る。また、膵がんのように腫瘍の密度が非常に疎である腫瘍では、線維成分が間質に造成しているため、診断の際に腫瘍細胞を同定しにくい場合があり、診断に苦慮することがある。そこで、腫瘍形成の際にみられる線維症を指標とすれば、膵がんの診断能力を向上させ得ると考えられる。したがって、線維芽細胞や筋線維芽細胞の分化を誘導し、線維化と密接に関連するＴＧＦ−β１を、抗体を用いて検出することにより線維症の診断を簡便に行えることが見込まれる。
　しかしながら、がんや線維症の診断に使用し得る程度に、特異性が高く、感度の良い抗体がないのが現状である。即ち、従来公知の抗ＴＧＦ−β１抗体（非特許文献１~５）のほとんどは、本来染色されるべきＴＧＦ−β１を発現している組織における染色性が低い、及び／又は活性型ＴＧＦ−β１だけでなく、潜在型ＴＧＦ−β１やＴＧＦ−β２、ＴＧＦ−β３等のオフターゲットタンパク質をも認識してしまうといった問題があり、いずれもがんや線維症の診断には不適である。 Hematoxylin-eosin (HE) staining is a simple and general method for diagnosing cancer, but this technique alone can not be used to diagnose cancer malignancy and prognosis, so a treatment method is used. When making a selection, diagnostic imaging such as CT is used in combination. Therefore, in order to evaluate the malignancy of cancer more easily, TGF-β1 which is deeply involved in cancer invasion / metastasis and immunosuppression is detected by immunostaining using an antibody, Western blot, FACS, etc. Methods have been tried since before.
On the other hand, there is fibrosis that correlates with the function of the organ, and when fibrosis is brought about as a result of chronic inflammation, for example, liver cirrhosis in the liver and lung fibrosis in the lungs, such as liver dysfunction and respiratory function decline Get into a serious condition. In addition, in a tumor such as pancreatic cancer where the density of the tumor is very low, since the fiber component is formed in the stroma, it may be difficult to identify a tumor cell at the time of diagnosis, which makes it difficult to diagnose. Sometimes. Therefore, it is considered that the diagnostic ability of pancreatic cancer can be improved if fibrosis observed at the time of tumor formation is used as an index. Therefore, it is expected that diagnosis of fibrosis can be conveniently performed by inducing differentiation of fibroblasts and myofibroblasts and detecting TGF-β1 closely associated with fibrosis using an antibody.
However, at present, there is no antibody with high specificity and high sensitivity to the extent that it can be used for diagnosis of cancer and fibrosis. That is, most of the conventionally known anti-TGF-β1 antibodies (non-patent documents 1 to 5) have low staining in tissues expressing TGF-β1 to be originally stained and / or active TGF-β1 In addition to this, there is also the problem of recognizing even an off-target protein such as latent TGF-.beta.1, TGF-.beta.2 or TGF-.beta.3, which are all unsuitable for diagnosis of cancer and fibrosis.
　従って、本発明の目的は、十分な感度があり、かつ活性型ＴＧＦ−β１か潜在型ＴＧＦ−β１及びＬＡＰのいずれか一方に特異的であり、他のＴＧＦ−βアイソフォーム等のオフターゲットタンパク質とも交差反応しない、特異性の高い抗ＴＧＦ−β１抗体を用い、がんや線維症の診断において有意義な情報を提供し得る新規手法を開発することである。 Thus, the object of the present invention is that it is sufficiently sensitive and specific for either active TGF-β1 or latent TGF-β1 and LAP, and an off-target protein such as another TGF-β isoform It is also to develop a new method that can provide meaningful information in diagnosis of cancer and fibrosis using highly specific anti-TGF-β1 antibody that does not cross-react either.
　本発明者は、株式会社ボナックとＡＶＮＯＶＡ社が共同開発した、活性型ＴＧＦ−β１を特異的に認識する新規モノクローナル抗体（抗体（１））と、潜在型ＴＧＦ−β１及びＬＡＰを特異的に認識する新規モノクローナル抗体（抗体（２））の提供を受け、それらを用いて大腸がん患者から採取した組織標本について免疫染色を実施した結果、これらの抗体が、市販の各種抗体と比較して、組織の種類（がん組織、正常腺組織、筋線維、線維芽細胞）を問わず感度（染色性）に優れること、並びに、がん組織及び非がん部における染色性において、抗体（１）と抗体（２）との間で差違がある（即ち、組織により活性型及び潜在型ＴＧＦ−β１の存在比が異なる）ことを見出し、一方の抗体を単独で用いるか、両抗体を組み合わせることによって、あるいは、従来のＨＥ染色をさらに組み合わせることによって、がんの悪性度（遠隔転移、周辺組織への浸潤など）や腫瘍随伴線維症を含む線維性疾患の診断が可能となることを明らかにした。
　本発明者は、これらの知見に基づいてさらに研究を重ねた結果、本発明を完成するに至った。 The present inventor specifically recognizes latent TGF-β1 and LAP specifically with a novel monoclonal antibody (antibody (1)) that specifically recognizes active TGF-β1, jointly developed by Bonak and AVNOVA. As a result of performing immunostaining on tissue specimens collected from colon cancer patients using these novel monoclonal antibodies (antibody (2)) and comparing these antibodies with various commercially available antibodies, Excellent sensitivity (stainability) regardless of the type of tissue (cancer tissue, normal gland tissue, muscle fibers, fibroblasts), and antibody (1) in the stainability in cancer tissue and non-cancerous part And there is a difference between antibody and antibody (2) (ie, the abundance ratio of active and latent TGF-β1 differs depending on the tissue), and either one antibody alone or both antibodies may be combined. The Or, by combining conventional HE staining, it is clear that diagnosis of fibrotic diseases including cancer malignancy (distant metastasis, invasion to surrounding tissues, etc.) and paraneoplastic fibrosis can be made. did.
As a result of further studies based on these findings, the present inventors have completed the present invention.
　即ち、本発明は以下の通りである。
［１］がんもしくは線維症患者、又はその疑いのある者由来の検体と、
　以下の（ａ１）~（ｆ１）の相補性決定領域（ＣＤＲ）：
（ａ１）配列番号１で表されるアミノ酸配列を含むＣＤＲ、
（ｂ１）配列番号２で表されるアミノ酸配列を含むＣＤＲ、
（ｃ１）配列番号３で表されるアミノ酸配列を含むＣＤＲ、
（ｄ１）配列番号４で表されるアミノ酸配列を含むＣＤＲ、
（ｅ１）配列番号５で表されるアミノ酸配列を含むＣＤＲ、及び
（ｆ１）配列番号６で表されるアミノ酸配列を含むＣＤＲ
を含み、活性型ＴＧＦ−β１に結合するが、潜在型ＴＧＦ−β１には結合しない第１のモノクローナル抗体、及び／又は
　以下の（ａ２）~（ｆ２）のＣＤＲ：
（ａ２）配列番号７で表されるアミノ酸配列を含むＣＤＲ、
（ｂ２）配列番号８で表されるアミノ酸配列を含むＣＤＲ、
（ｃ２）配列番号９で表されるアミノ酸配列を含むＣＤＲ、
（ｄ２）配列番号１０で表されるアミノ酸配列を含むＣＤＲ、
（ｅ２）配列番号１１で表されるアミノ酸配列を含むＣＤＲ、及び
（ｆ２）配列番号１２で表されるアミノ酸配列を含むＣＤＲ
を含み、潜在型ＴＧＦ−β１及びＬＡＰに結合するが、活性型ＴＧＦ−β１には結合しない第２のモノクローナル抗体
とを接触させ、該検体中の活性型ＴＧＦ−β１及び／又は潜在型ＴＧＦ−β１もしくはＬＡＰを特異的に検出することを特徴とする、がんもしくは線維症の診断、又はがんの悪性度もしくは線維化の進行度の診断のための検査方法。
［２］第１のモノクローナル抗体の重鎖可変領域に含まれるＣＤＲが上記（ａ１）、（ｂ１）及び（ｃ１）であり、軽鎖可変領域に含まれるＣＤＲが上記（ｄ１）、（ｅ１）及び（ｆ１）であり、
　第２のモノクローナル抗体の重鎖可変領域に含まれるＣＤＲが上記（ａ２）、（ｂ２）及び（ｃ２）であり、軽鎖可変領域に含まれるＣＤＲが上記（ｄ２）、（ｅ２）及び（ｆ２）である、
［１］に記載の方法。
［３］第１のモノクローナル抗体の重鎖可変領域に含まれるＣＤＲ１、ＣＤＲ２及びＣＤＲ３がそれぞれ上記（ａ１）、（ｂ１）及び（ｃ１）であり、軽鎖可変領域に含まれるＣＤＲ１、ＣＤＲ２及びＣＤＲ３がそれぞれ上記（ｄ１）、（ｅ１）及び（ｆ１）であり、
　第２のモノクローナル抗体の重鎖可変領域に含まれるＣＤＲ１、ＣＤＲ２及びＣＤＲ３がそれぞれ上記（ａ２）、（ｂ２）及び（ｃ２）であり、軽鎖可変領域に含まれるＣＤＲ１、ＣＤＲ２及びＣＤＲ３がそれぞれ上記（ｄ２）、（ｅ２）及び（ｆ２）である、
［１］に記載の方法。
［４］第１のモノクローナル抗体が、
（Ｘ１）配列番号１３で表されるアミノ酸配列を含む重鎖可変領域、及び
（Ｙ１）配列番号１４で表されるアミノ酸配列を含む軽鎖可変領域
を含み、
　第２のモノクローナル抗体が、
（Ｘ２）配列番号１５で表されるアミノ酸配列を含む重鎖可変領域、及び
（Ｙ２）配列番号１６で表されるアミノ酸配列を含む軽鎖可変領域
を含む、［１］に記載の方法。
［５］第１のモノクローナル抗体が、［１］~［４］のいずれかに記載の第１のモノクローナル抗体と競合的に活性型ＴＧＦ−β１に結合し、かつ潜在型ＴＧＦ−β１には結合しない抗体であり、
　第２のモノクローナル抗体が、［１］~［４］のいずれかに記載の第２のモノクローナル抗体と競合的に潜在型ＴＧＦ−β１もしくはＬＡＰに結合し、かつ活性型ＴＧＦ−β１には結合しない抗体である、［１］~［４］のいずれかに記載の方法。
［６］検体が生検組織、体液又はエクソソームである、［１］~［５］のいずれかに記載の方法。
［７］検出法が、免疫組織染色、ＥＬＩＳＡ、ウェスタンブロット、ＦＡＣＳ及びＭＡＣＳからなる群より選択される、［１］~［６］のいずれかに記載の方法。
［８］以下の（ａ１）~（ｆ１）の相補性決定領域（ＣＤＲ）：
（ａ１）配列番号１で表されるアミノ酸配列を含むＣＤＲ、
（ｂ１）配列番号２で表されるアミノ酸配列を含むＣＤＲ、
（ｃ１）配列番号３で表されるアミノ酸配列を含むＣＤＲ、
（ｄ１）配列番号４で表されるアミノ酸配列を含むＣＤＲ、
（ｅ１）配列番号５で表されるアミノ酸配列を含むＣＤＲ、及び
（ｆ１）配列番号６で表されるアミノ酸配列を含むＣＤＲ
を含み、活性型ＴＧＦ−β１に結合するが、潜在型ＴＧＦ−β１には結合しない第１のモノクローナル抗体、及び
　以下の（ａ２）~（ｆ２）のＣＤＲ：
（ａ２）配列番号７で表されるアミノ酸配列を含むＣＤＲ、
（ｂ２）配列番号８で表されるアミノ酸配列を含むＣＤＲ、
（ｃ２）配列番号９で表されるアミノ酸配列を含むＣＤＲ、
（ｄ２）配列番号１０で表されるアミノ酸配列を含むＣＤＲ、
（ｅ２）配列番号１１で表されるアミノ酸配列を含むＣＤＲ、及び
（ｆ２）配列番号１２で表されるアミノ酸配列を含むＣＤＲ
を含み、潜在型ＴＧＦ−β１及びＬＡＰに結合するが、活性型ＴＧＦ−β１には結合しない第２のモノクローナル抗体
を含んでなる、がんもしくは線維症の診断、又はがんの悪性度もしくは線維化の進行度の診断用キット。 That is, the present invention is as follows.
[1] Samples from patients with cancer or fibrosis, or those suspected of:
The following (a1) to (f1) complementarity determining regions (CDRs):
(A1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 1,
(B1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 2,
(C1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 3,
(D1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 4,
(E1) CDR comprising the amino acid sequence represented by SEQ ID NO: 5, and (f1) CDR comprising the amino acid sequence represented by SEQ ID NO: 6
A first monoclonal antibody that binds to active TGF-β1 but not to latent TGF-β1, and / or CDRs of (a2) to (f2) below:
(A2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 7,
(B2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 8,
(C2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 9,
(D2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 10,
(E2) CDR comprising the amino acid sequence represented by SEQ ID NO: 11 and (f2) CDR comprising the amino acid sequence represented by SEQ ID NO: 12
Contact with a second monoclonal antibody that contains latent TGF-.beta.1 and LAP but does not bind to active TGF-.beta.1, active TGF-.beta.1 in the sample and / or latent TGF-.beta. A test method for diagnosing cancer or fibrosis, or diagnosing cancer malignancy or progression of fibrosis, which is characterized by specifically detecting β1 or LAP.
[2] The CDRs contained in the heavy chain variable region of the first monoclonal antibody are the above (a1), (b1) and (c1), and the CDRs contained in the light chain variable region are the above (d1), (e1) And (f1),
The CDRs contained in the heavy chain variable region of the second monoclonal antibody are the above (a2), (b2) and (c2), and the CDRs contained in the light chain variable region are the above (d2), (e2) and (f2) ),
The method described in [1].
[3] The CDR1, CDR2 and CDR3 contained in the heavy chain variable region of the first monoclonal antibody are the above (a1), (b1) and (c1) respectively, and the CDR1, CDR2 and CDR3 contained in the light chain variable region Are the above (d1), (e1) and (f1) respectively
The CDR1, CDR2 and CDR3 contained in the heavy chain variable region of the second monoclonal antibody are the above (a2), (b2) and (c2) respectively, and the CDR1, CDR2 and CDR3 contained in the light chain variable region are each mentioned above (D2), (e2) and (f2),
The method described in [1].
[4] The first monoclonal antibody is
(X1) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13; and (Y1) a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14;
The second monoclonal antibody is
(X2) The method according to [1], comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16 (Y2).
[5] The first monoclonal antibody binds to active TGF-β1 competitively with the first monoclonal antibody according to any of [1] to [4], and binds to latent TGF-β1 Not an antibody,
The second monoclonal antibody binds to latent TGF-β1 or LAP in competition with the second monoclonal antibody according to any of [1] to [4], and does not bind to active TGF-β1. The method according to any one of [1] to [4], which is an antibody.
[6] The method according to any one of [1] to [5], wherein the sample is a biopsy tissue, a body fluid or an exosome.
[7] The method according to any one of [1] to [6], wherein the detection method is selected from the group consisting of immunohistological staining, ELISA, western blot, FACS and MACS.
[8] The following (a1) to (f1) complementarity determining regions (CDRs):
(A1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 1,
(B1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 2,
(C1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 3,
(D1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 4,
(E1) CDR comprising the amino acid sequence represented by SEQ ID NO: 5, and (f1) CDR comprising the amino acid sequence represented by SEQ ID NO: 6
And a first monoclonal antibody that binds to active TGF-β1 but not latent TGF-β1, and the CDRs of (a2) to (f2) below:
(A2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 7,
(B2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 8,
(C2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 9,
(D2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 10,
(E2) CDR comprising the amino acid sequence represented by SEQ ID NO: 11 and (f2) CDR comprising the amino acid sequence represented by SEQ ID NO: 12
Diagnosis of cancer or fibrosis, or cancer grade or fiber comprising a second monoclonal antibody comprising: TGF-.beta.1 and LAP but binding to latent TGF-.beta.1 but not to active TGF-.beta.1 Diagnostic kit for
　本発明の抗体は、組織の種類を問わず活性型ＴＧＦ−β１又は潜在型ＴＧＦ−β１／ＬＡＰを感度よく分別して検出することができるので、該２種類の抗体を用いた病理組織上の局在解析により、腫瘍の悪性度（遠隔転移しやすいか、周辺組織に浸潤しやすいか）の判断、治療薬の反応の程度を反映するかなどの評価が可能になる。また、ＥＬＩＳＡ等により血中ＴＧＦ−β１の濃度を測定し、活性型と潜在型の発現比較解析により悪性度の判断が可能となる。さらに、ＦＡＣＳやＭＡＣＳを用いたエクソソーム解析により、早期がん診断、予後予測が可能となる。
　また、本発明によれば、腫瘍に伴う線維症や全身の線維症の診断が可能となる。 The antibody of the present invention can detect active TGF-β1 or latent TGF-β1 / LAP with high sensitivity regardless of the type of tissue. In-situ analysis makes it possible to evaluate the degree of malignancy of a tumor (whether it is likely to metastasize or to invade surrounding tissues), and to evaluate whether it reflects the degree of response of therapeutic agents. In addition, the concentration of TGF-β1 in blood is measured by ELISA or the like, and expression comparison analysis of active and latent forms enables judgment of malignancy. Furthermore, exosome analysis using FACS or MACS enables early cancer diagnosis and prognosis.
Furthermore, according to the present invention, it is possible to diagnose fibrosis associated with a tumor and fibrosis in the whole body.
市販の各種抗ＴＧＦ−β１抗体を用いた大腸がん組織標本の免疫染色像を示す図である。It is a figure which shows the immunostaining image of colon cancer tissue sample using various marketed anti-TGF- (beta) 1 antibodies. 本発明の抗潜在型ＴＧＦ−β１抗体／ＬＡＰ抗体及び市販の各種抗ＴＧＦ−β１抗体を用いた大腸がん組織標本の免疫染色像を示す図である。It is a figure which shows the immunostaining image of the colon cancer tissue sample using the anti-latent type | mold TGF- (beta) 1 antibody / LAP antibody of this invention, and various commercially available anti-TGF- (beta) 1 antibodies. 本発明の抗潜在型ＴＧＦ−β１抗体／ＬＡＰ抗体及び抗活性型ＴＧＦ−β１抗体を用いた大腸がん組織標本の免疫染色像を示す図である。上から順に、ＨＥ染色像、抗活性型ＴＧＦ−β１抗体を一次抗体として用いた免疫染色像、抗潜在型ＴＧＦ−β１／ＬＡＰ抗体を一次抗体として用いた免疫染色像、ネガティブコントロール（一次抗体なし）を示す。It is a figure which shows the immunostaining image of the colon cancer tissue sample using the anti-latent type TGF- (beta) 1 antibody / LAP antibody of this invention, and anti-activation type TGF- (beta) 1 antibody. From top to bottom, HE staining image, immunostaining image using anti-active TGF-β1 antibody as primary antibody, immunostaining image using anti-latent TGF-β1 / LAP antibody as primary antibody, negative control (no primary antibody ). 本発明の抗潜在型ＴＧＦ−β１抗体／ＬＡＰ抗体（２Ｆ１０）及び抗活性型ＴＧＦ−β１抗体（２Ｈ４及び４Ｄ１０）を用いた大腸がん組織標本の免疫染色像を示す図である。ＨＥはヘマトキシリン−エオシン染色像、ＮＣはネガティブコントロール（一次抗体なし）を示す。It is a figure which shows the immunostaining image of the colon cancer tissue sample using the anti-latent type | mold TGF- (beta) 1 antibody / LAP antibody (2F10) and anti-activation type TGF- (beta) 1 antibody (2H4 and 4D10) of this invention. HE shows hematoxylin-eosin stained image, and NC shows negative control (without primary antibody).
［Ｉ］定義
　本明細書において、アミノ酸、（ポリ）ペプチド、（ポリ）ヌクレオチドなどの略号による表示は、ＩＵＰＡＣ−ＩＵＢの規定〔ＩＵＰＡＣ−ＩＵＢ　Ｃｏｍｍｕｎｉｃａｔｉｏｎ　ｏｎ　Ｂｉｏｌｏｇｉｃａｌ　Ｎｏｍｅｎｃｌａｔｕｒｅ，Ｅｕｒ．Ｊ．Ｂｉｏｃｈｅｍ．，１３８：９（１９８４）〕、「塩基配列又はアミノ酸配列を含む明細書等の作成のためのガイドライン」（日本国特許庁編）、及び当該分野における慣用記号に従う。 [I] Definitions In the present specification, designations by abbreviations such as amino acids, (poly) peptides, (poly) nucleotides, etc. are as defined in IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem. 138: 9 (1984)], “Guidelines for preparation of a specification including a nucleotide sequence or an amino acid sequence” (edited by the Japan Patent Office), and conventional symbols in the art.
　本明細書において「遺伝子」又は「ＤＮＡ」とは、２本鎖ＤＮＡのみならず、それを構成するセンス鎖及びアンチセンス鎖といった各１本鎖ＤＮＡを包含する趣旨で用いられる。またその長さによって特に制限されるものではない。従って、本明細書において遺伝子（ＤＮＡ）とは、特に言及しない限り、ヒトゲノムＤＮＡを含む２本鎖ＤＮＡ、ｃＤＮＡを含む１本鎖ＤＮＡ（正鎖）及び該正鎖と相補的な配列を有する１本鎖ＤＮＡ（相補鎖）、並びにこれらの断片のいずれもが含まれる。 In the present specification, “gene” or “DNA” is used to include not only double-stranded DNA but also single-stranded DNAs such as the sense strand and the antisense strand that constitute it. Also, the length is not particularly limited. Therefore, unless otherwise stated, a gene (DNA) in the present specification has a double-stranded DNA containing human genomic DNA, a single-stranded DNA (positive strand) containing cDNA, and a sequence complementary to the positive strand 1 Included are single stranded DNA (complementary strand), as well as any of these fragments.
　本明細書において「ＴＧＦ−β１遺伝子」とは、そのｃＤＮＡ配列がＧｅｎＢａｎｋにａｃｃｅｓｓｉｏｎ　Ｎｏ．ＮＭ＿０００６６０として登録されているヒトＴＧＦ−β１遺伝子（ＤＮＡ）、その天然の変異体又は多型バリアントを意味する。そのような変異体又は多型バリアントとしては、例えば、ＮＣＢＩから入手可能なＳＮＰデータベースに登録されたものが挙げられる。 As used herein, the term "TGF-β1 gene" refers to the cDNA sequence of the accession no. It means the human TGF-β1 gene (DNA) registered as NM — 000660, its natural variant or polymorphic variant. Such variants or polymorphic variants include, for example, those registered in the SNP database available from NCBI.
　本明細書において「ＴＧＦ−β１タンパク質」又は単に「ＴＧＦ−β１」といった用語は、そのアミノ酸配列がＧｅｎＢａｎｋにａｃｃｅｓｓｉｏｎ　Ｎｏ．ＮＰ＿０００６５１として登録されているヒトＴＧＦ−β１タンパク質（該配列中、１−２９位がシグナルペプチド、３０−２７８位がＬＡＰ及び２７９−３９０位が成熟ＴＧＦ−β１である）、又は上記した天然の変異体又は多型バリアントＤＮＡによりコードされるタンパク質を意味する。 As used herein, the terms “TGF-β1 protein” or simply “TGF-β1” have their amino acid sequences described in GenBank accession no. Human TGF-β1 protein registered as NP_000651 (in the sequence, the signal peptide at position 1-29, LAP at position 30-278 and mature TGF-β1 at position 279), or the natural mutation described above A protein encoded by a body or polymorphic variant DNA is meant.
　本明細書において「潜在型ＴＧＦ−β１」とは、成熟ＴＧＦ−β１のホモダイマー及び非共有結合によりそれと結合したＬＡＰのホモダイマー、任意でさらにＬＴＢＰからなる不活性型のＴＧＦ−β１を意味する。「活性型ＴＧＦ−β１」とは潜在型ＴＧＦ−β１から遊離した成熟ＴＧＦ−β１のホモダイマーを意味する。 As used herein, “latent TGF-β1” refers to a mature TGF-β1 homodimer and an inactive form of TGF-β1 consisting of a homodimer of LAP bound thereto noncovalently, optionally further comprising LTBP. "Active TGF-β1" means a homodimer of mature TGF-β1 released from latent TGF-β1.
　本明細書でいう「抗体」には、ポリクローナル抗体、モノクローナル抗体、キメラ抗体、一本鎖抗体、又はＦａｂフラグメントなどのように抗原結合性を有する上記抗体の一部が包含される。 The term "antibody" as used herein includes a part of the above-mentioned antibody having antigen binding ability, such as polyclonal antibody, monoclonal antibody, chimeric antibody, single chain antibody, or Fab fragment.
　本明細書においてエピトープとは、抗体が結合する抗原の領域である。ある種の実施形態では、免疫グロブリンに特異的に結合し得る抗原の任意の部位を含む。抗原決定基は、分子の化学的に活性な表面群、例えばアミノ酸、糖側鎖、ホスホリル基又はスルホニル基を含み、ある種の実施形態では、特異的な三次元構造特徴及び／又は特異的な荷電特徴を有してよい。ある種の実施形態では、抗体は、タンパク質及び／又は巨大分子の複雑な混合物において、この抗体が標的抗原を優先的に認識する場合、抗原に特異的に結合するということができる。 As used herein, an epitope is a region of an antigen to which an antibody binds. In certain embodiments, it comprises any portion of an antigen that can specifically bind to an immunoglobulin. An antigenic determinant comprises a chemically active surface group of molecules such as amino acids, sugar side chains, phosphoryl groups or sulfonyl groups, and in certain embodiments, specific three dimensional structural features and / or specific It may have charged features. In certain embodiments, an antibody can be said to specifically bind an antigen if it preferentially recognizes a target antigen in a complex mixture of proteins and / or macromolecules.
抗体の構造
　抗体分子の基本構造は、各クラス共通で、分子量５−７万の重鎖と２−３万の軽鎖から構成される（免疫学イラストレイテッド（Ｉ．Ｒｏｉｔｔ，Ｊ．Ｂｒｏｓｔｏｆｆ，Ｄ．Ｍａｌｅ編））。重鎖は、通常約４４０個のアミノ酸を含むポリペプチド鎖からなり、クラスごとに特徴的な構造をもち、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥに対応してγ、μ、α、δ、ε鎖とよばれる。さらにＩｇＧには、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４が存在し、それぞれγ１、γ２、γ３、γ４とよばれている。軽鎖は、通常約２２０個のアミノ酸を含むポリペプチド鎖からなり、Ｌ型とＫ型の２種が知られており、それぞれλ、κ鎖とよばれる。抗体分子の基本構造のペプチド構成は、それぞれ相同な２本の重鎖及び２本の軽鎖が、ジスルフィド結合（Ｓ−Ｓ結合）及び非共有結合によって結合され、分子量１５−１９万である。２種の軽鎖は、どの重鎖とも対をなすことができる。個々の抗体分子は、常に同一の軽鎖２本と同一の重鎖２本からできている。 Structure of antibody The basic structure of the antibody molecule is common to all classes and is composed of a heavy chain having a molecular weight of 5 to 70,000 and a light chain having a molecular weight of 30,000 (I. Roitt, J. Brostoff, D. Male edition)). The heavy chain usually consists of a polypeptide chain containing about 440 amino acids, and has a distinctive structure for each class, corresponding to IgG, IgM, IgA, IgD, and IgE, γ, μ, α, δ, ε It is called a chain. Furthermore, IgG1, IgG2, IgG3 and IgG4 are present as IgG, and they are called γ1, γ2, γ3 and γ4, respectively. The light chain is usually composed of a polypeptide chain containing about 220 amino acids, and two types, L-type and K-type, are known, which are called λ and 鎖 chains, respectively. The peptide composition of the basic structure of the antibody molecule is such that two heavy chains and two light chains, which are homologous to each other, are linked by disulfide bond (S-S bond) and non-covalent bond, and have a molecular weight of 15-190,000. The two light chains can be paired with any heavy chain. Each antibody molecule is always made up of two identical light chains and two identical heavy chains.
　鎖内Ｓ−Ｓ結合は、重鎖に４つ（μ、ε鎖には５つ）、軽鎖には２つあって、アミノ酸１００−１１０残基ごとに１つのループを形成し、この立体構造は各ループ間で類似していて、構造単位あるいはドメインとよばれる。重鎖、軽鎖ともにＮ末端に位置するドメインは、同種動物の同一クラス（サブクラス）からの標品であっても、そのアミノ酸配列が一定せず、可変領域（Ｖ領域）とよばれている（重鎖可変領域ドメインはＶＨ、軽鎖可変領域ドメインはＶＬと表される）。これよりＣ末端側のアミノ酸配列は、各クラスあるいはサブクラスごとにほぼ一定で定常領域（Ｃ領域）とよばれている（各ドメインは、それぞれ、ＣＨ１、ＣＨ２、ＣＨ３あるいはＣＬと表される）。 There are four intrachain SS bonds (μ, five in the ε chain) and two in the light chain, forming a loop for every 100-110 amino acid residues, The structures are similar between each loop and are called structural units or domains. Domains located at the N-terminus of both heavy chain and light chain are referred to as variable regions (V regions), whose amino acid sequences are not constant even if they are preparations from the same class (subclass) of the same animal (The heavy chain variable domain is designated VH and the light chain variable domain is designated VL). The amino acid sequence at the C-terminal side from this is almost constant for each class or subclass, and is called a constant region (C region) (each domain is represented as CH1, CH2, CH3 or CL, respectively).
　抗体の抗原決定部位はＶＨ及びＶＬによって構成され、結合の特異性はこの部位のアミノ酸配列によっている。一方、補体や各種細胞との結合といった生物学的活性は各クラスＩｇのＣ領域の構造の差を反映している。軽鎖と重鎖の可変領域の可変性は、どちらの鎖にも存在する３つの小さな超可変領域にほぼ限られることが分かっており、これらの領域を相補性決定領域（ＣＤＲ）と呼んでいる。可変領域のうち、ＣＤＲを除く部分はフレームワーク領域（ＦＲ）とよばれ、比較的一定である。フレームワーク領域は、βシートコンフォメーションを採用しており、ＣＤＲはβシート構造を接続するループを形成することができる。各鎖におけるＣＤＲは、フレームワーク領域によりそれらの三次元構造が保持され、他の鎖からのＣＤＲと共に抗原結合部位を形成する。 The antigenic determination site of the antibody is constituted by VH and VL, and the specificity of binding depends on the amino acid sequence of this site. On the other hand, biological activities such as complement and binding to various cells reflect differences in the structure of the C region of each class Ig. It has been found that the variability of the light chain and heavy chain variable regions is substantially limited to the three small hypervariable regions present in either chain, and these regions are referred to as complementarity determining regions (CDRs) There is. The part of the variable region other than the CDR is called a framework region (FR) and is relatively constant. The framework regions adopt a beta sheet conformation, and the CDRs can form loops connecting beta sheet structures. The CDRs in each chain retain their three dimensional structure by the framework regions and form an antigen binding site with the CDRs from the other chain.
　ＣＤＲを同定するためのいくつかのナンバリングシステムが一般に使用されている。
Ｋａｂａｔ定義は、配列変化性に基づき、Ｃｈｏｔｈｉａ定義は、構造ループ領域の位置に基づく。ＡｂＭ定義は、Ｋａｂａｔ及びＣｈｏｔｈｉａアプローチの間の折衷である。軽鎖・重鎖の可変領域のＣＤＲは、Ｋａｂａｔ、Ｃｈｏｔｈｉａ又はＡｂＭアルゴリズムにしたがって、境界を示される　（Ｍａｒｔｉｎ　ｅｔ　ａｌ．（１９８９）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８６：９２６８−９２７２；Ｍａｒｔｉｎ　ｅｔ　ａｌ．（１９９１）Ｍｅｔｈｏｄｓ　Ｅｎｚｙｍｏｌ．２０３：１２１−１５３；Ｐｅｄｅｒｓｅｎ　ｅｔ　ａｌ．（１９９２）Ｉｍｍｕｎｏｍｅｔｈｏｄｓ　１：１２６；及びＲｅｅｓ　ｅｔ　ａｌ．（１９９６）Ｉｎ　Ｓｔｅｒｎｂｅｒｇ　Ｍ．Ｊ．Ｅ．（ｅｄ．），Ｐｒｏｔｅｉｎ　Ｓｔｒｕｃｔｕｒｅ　Ｐｒｅｄｉｃｔｉｏｎ，Ｏｘｆｏｒｄ　Ｕｎｉｖｅｒｓｉｔｙ　Ｐｒｅｓｓ，Ｏｘｆｏｒｄ，ｐｐ．１４１−１７２）。 Several numbering systems for identifying CDRs are commonly used.
The Kabat definition is based on sequence variability and the Chothia definition is based on the position of the structural loop region. AbM definition is a compromise between the Kabat and Chothia approaches. The CDRs of the light chain and heavy chain variable regions are demarcated according to the Kabat, Chothia or AbM algorithm (Martin et al. (1989) Proc. Natl. Acad. Sci. USA 86: 9268-9272; Martin et al. (1991) Methods Enzymol. 203: 121-153; Pedersen et al. (1992) Immunomethods 1: 126; and Rees et al. (1996) In Sternberg M. J. E. (ed.), Protein Structure Prediction. , Oxford University Press, Oxford, pp. 141-172).
　本発明の抗体のＣＤＲは、該抗体の重鎖及び軽鎖の可変領域（ＶＨ及びＶＬ）のヌクレオチド配列を、公共のＣＤＲ決定ソフトウェア（ｈｔｔｐ：／／ｗｗｗ．ａｂｙｓｉｓ．ｏｒｇ及びｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｉｇｂｌａｓｔ／ｉｇｂｌａｓｔ．ｃｇｉ）を用いて解析することにより同定されるＣＤＲであると定義づけられる。
　本発明の第１のモノクローナル抗体の場合、重鎖可変領域のＣＤＲは、配列番号：１３で表されるアミノ酸配列中アミノ酸番号２６~３２（ＣＤＲ１−Ｈ）、５２~５７（ＣＤＲ２−Ｈ）及び９９~１０９（ＣＤＲ３−Ｈ）であり、軽鎖可変領域のＣＤＲは、配列番号：１４で表されるアミノ酸配列中アミノ酸番号２４~４０（ＣＤＲ１−Ｌ）、５２~５７（ＣＤＲ２−Ｌ）及び９９~１０２（ＣＤＲ３−Ｌ）である。本発明の第２のモノクローナル抗体の場合、重鎖可変領域のＣＤＲは、配列番号：１５で表されるアミノ酸配列中アミノ酸番号２６~３２（ＣＤＲ１−Ｈ）、５２~５７（ＣＤＲ２−Ｈ）及び９９~１０３（ＣＤＲ３−Ｈ）であり、軽鎖可変領域のＣＤＲは、配列番号：１６で表されるアミノ酸配列中アミノ酸番号２４~３４（ＣＤＲ１−Ｌ）、５０~５６（ＣＤＲ２−Ｌ）及び８９~９７（ＣＤＲ３−Ｌ）である。 The CDRs of the antibody of the present invention comprise the nucleotide sequences of the variable regions (VH and VL) of the heavy chain and light chain of the antibody, as well as public CDR determination software (http://www.abysis.org and http: // www It is defined as a CDR identified by analysis using .ncbi.nlm.nih.gov / igblast / igblast.cgi).
In the case of the first monoclonal antibody of the present invention, the CDRs of the heavy chain variable region are amino acids 26-32 (CDR1-H), 52-57 (CDR2-H), and the like in the amino acid sequence represented by SEQ ID NO: 13 99 to 109 (CDR 3-H), and the CDRs of the light chain variable region are amino acid Nos. 24 to 40 (CDR 1-L), 52 to 57 (CDR 2-L), and the amino acid sequences represented by SEQ ID NO: 14 99 to 102 (CDR 3-L). In the case of the second monoclonal antibody of the present invention, the CDRs of the heavy chain variable region are amino acid Nos. 26-32 (CDR1-H), 52-57 (CDR2-H) in the amino acid sequence represented by SEQ ID NO: 15 and 99 to 103 (CDR 3-H), and the CDRs of the light chain variable region are amino acid numbers 24 to 34 (CDR 1-L), 50 to 56 (CDR 2-L) and the amino acid sequences represented by SEQ ID NO: 16 89 to 97 (CDR 3-L).
抗体の結合アッセイ
　抗体結合性の確認は任意の周知のアッセイ方法、例えば、直接及び間接サンドイッチアッセイ、フローサイトメトリー及び免疫沈降アッセイ等で行うことができる（Ｚｏｌａ，Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ：Ａ　Ｍａｎｕａｌ　ｏｆ　Ｔｅｃｈｎｉｑｕｅｓ，（ＣＲＣ　Ｐｒｅｓｓ，Ｉｎｃ．１９８７）ｐｐ．１４７−１５８）。本発明において、抗ＴＧＦ−β１抗体の活性型ＴＧＦ−β１又は潜在型ＴＧＦ−β１／ＬＡＰポリペプチドとの結合は、例えば、以下の方法に従って測定することができる。 Antibody Binding Assay The confirmation of antibody binding can be carried out by any known assay method, such as direct and indirect sandwich assays, flow cytometry and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, ( CRC Press, Inc. 1987) pp. 147-158). In the present invention, the binding of an anti-TGF-β1 antibody to active TGF-β1 or latent TGF-β1 / LAP polypeptide can be measured, for example, according to the following method.
　典型的な方法として、ヒトＴＧＦ−β１／ＬＡＰポリペプチド（抗原）を固相に吸着させ、抗原抗体反応や酵素反応に関与しないタンパク質（スキムミルク、アルブミン等）でブロッキングした後、抗ＴＧＦ−β１モノクローナル抗体（被検抗体）を固相に接触させてインキュベートし、Ｂ／Ｆ分離により未反応の抗体を除いた後、被検抗体と特異的に反応する標識化二次抗体（抗マウスＩｇＧ等）を作用させて、固相上の標識量を測定する方法が挙げられる。固相としては、例えば、アガロース、デキストラン、セルロースなどの不溶性多糖類、プラスチック、ポリスチレン、ポリアクリルアミド、シリコン等の合成樹脂（チューブ、マイクロプレート等）、あるいはガラス（ビーズ、チューブ等）などを用いることができる。標識剤としては、例えば、放射性同位元素、酵素、蛍光物質、発光物質などが用いられる。放射性同位元素としては、例えば、〔１２５Ｉ〕、〔１３１Ｉ〕、〔３Ｈ〕、〔１４Ｃ〕などが用いられる。上記酵素としては、安定で比活性の大きなものが好ましく、例えば、β−ガラクトシダーゼ、β−グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素などが用いられる。蛍光物質としては、例えば、フルオレスカミン、フルオレセインイソチオシアネートなどが用いられる。発光物質としては、例えば、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなどが用いられる。 As a typical method, human TGF-β1 / LAP polypeptide (antigen) is adsorbed on a solid phase and blocked with a protein (skimmed milk, albumin, etc.) not involved in antigen-antibody reaction or enzyme reaction, and then anti-TGF-β1 monoclonal An antibody (test antibody) is brought into contact with a solid phase and incubated, and after removing an unreacted antibody by B / F separation, a labeled secondary antibody (anti-mouse IgG etc.) specifically reacting with the test antibody Methods to measure the amount of labeling on the solid phase by As the solid phase, for example, insoluble polysaccharides such as agarose, dextran and cellulose, plastics, polystyrene, polyacrylamide, synthetic resins such as silicon (tubes, microplates, etc.) or glass (beads, tubes etc.) etc. Can. As the labeling agent, for example, radioactive isotopes, enzymes, fluorescent substances, luminescent substances and the like are used. As the radioactive isotope, for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used. As the above-mentioned enzyme, one which is stable and has a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the light-emitting substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
競合アッセイ
　本発明の抗ＴＧＦ−β１抗体の結合定数（Ｋａ）の測定や、既に得られた本発明の抗体と競合的に活性型又は潜在型ＴＧＦ−β１に結合する本発明の他の抗体の同定には、競合ＥＬＩＳＡなどの競合アッセイを用いることができる。競合アッセイは、上記の抗原固定化固相を用いる結合アッセイにおいて、固相と被検抗体との反応系に遊離の抗原もしくは既知抗体を共存させることにより実施される。例えば、既知濃度の被検抗体液、並びに種々の濃度の抗原を該被検抗体液に加えた混合液を抗原固定化固相に接触させてインキュベートし、固相上の標識量をそれぞれ測定する。各遊離抗原濃度における測定値からスキャッチャード解析を行い、グラフの傾きを結合定数として算出することができる。一方、抗原固定化固相に対して、標識した既知抗体（本発明の抗体）と、種々の濃度の被検抗体とを反応させ、固相上の標識量を濃度依存的に減少させた被検抗体を選択することにより、本発明の抗体と競合的に活性型又は潜在型ＴＧＦ−β１に結合する抗体を同定することができる。 Competition Assay Measurement of the binding constant (Ka) of the anti-TGF-β1 antibody of the present invention, and other antibodies of the present invention that bind to active or latent TGF-β1 competitively with the previously obtained antibody of the present invention A competitive assay such as a competitive ELISA can be used for identification. The competition assay is carried out by causing free antigen or known antibody to coexist in the reaction system of the solid phase and the test antibody in the binding assay using the antigen-immobilized solid phase described above. For example, a test antibody solution of known concentration and a mixed solution obtained by adding antigens of various concentrations to the test antibody solution are brought into contact with an antigen-immobilized solid phase and incubated, and the labeled amount on the solid phase is measured respectively . Scatchard analysis can be performed from the measured values at each free antigen concentration, and the slope of the graph can be calculated as a binding constant. On the other hand, an antigen-immobilized solid phase is reacted with a labeled known antibody (the antibody of the present invention) and test antibodies of various concentrations to reduce the labeled amount on the solid phase in a concentration-dependent manner. By selecting a test antibody, it is possible to identify an antibody that binds to active or latent TGF-β1 competitively with the antibody of the present invention.
［ＩＩ］本発明の検査方法
（ＩＩ−１）本発明の抗体
　本発明は、活性型ＴＧＦ−β１に結合するが、潜在型ＴＧＦ−β１には結合しない第１のモノクローナル抗体（以下、「本発明の抗体（１）」ともいう）、及び／又は、潜在型ＴＧＦ−β１もしくはＬＡＰに結合するが、活性型ＴＧＦ−β１には結合しない第２のモノクローナル抗体（以下、「本発明の抗体（２）」ともいう）を用いることを特徴とする、がんもしくは線維症の診断、又はがんの悪性度もしくは線維化の進行度の診断のための検査方法（以下、「本発明の検査方法」ともいう）を提供する。本発明の抗体（１）と本発明の抗体（２）とを包括して「本発明の抗体」という。本発明の抗体はさらに、該抗体により認識されるエピトープから予測されるいかなるオフターゲットタンパク質を認識しないことにより特徴づけられる。 [II] Test Method of the Present Invention (II-1) Antibody of the Present Invention The present invention provides a first monoclonal antibody that binds to active TGF-β1 but does not bind to latent TGF-β1 (hereinafter referred to as “the present monoclonal antibody A second monoclonal antibody (hereinafter referred to as “the antibody according to the present invention (hereinafter referred to as“ the antibody according to the invention (hereinafter referred to as “the antibody according to the invention (hereinafter referred to as“ antibody according to the invention 2) “test method for diagnosis of cancer or fibrosis, or diagnosis of cancer malignancy or progression of fibrosis (hereinafter referred to as“ test method according to the present invention, ”) Also referred to as The antibody (1) of the present invention and the antibody (2) of the present invention are collectively referred to as "the antibody of the present invention". The antibodies of the invention are further characterized by not recognizing any off-target proteins predicted from the epitopes recognized by the antibodies.
　本発明の抗体（１）は、（ｉ）以下の（ａ１）~（ｆ１）の相補性決定領域（ＣＤＲ）：
（ａ１）Ｇｌｙ　Ｔｙｒ　Ｔｈｒ　Ｐｈｅ　Ｓｅｒ　Ａｓｎ　Ｔｙｒで表されるアミノ酸配列（配列番号１）を含むＣＤＲ、
（ｂ１）Ｔｙｒ　Ｐｒｏ　Ｇｌｙ　Ａｓｎ　Ｓｅｒ　Ａｓｐで表されるアミノ酸配列（配列番号２）を含むＣＤＲ、
（ｃ１）Ｔｙｒ　Ｓｅｒ　Ａｓｎ　Ｔｙｒ　Ｇｌｕ　Ａｌａ　Ｇｌｙ　Ａｌａ　Ｍｅｔ　Ａｓｐ　Ｔｙｒで表されるアミノ酸配列（配列番号３）を含むＣＤＲ、
（ｄ１）Ｌｙｓ　Ｓｅｒ　Ｓｅｒ　Ｇｌｎ　Ｓｅｒ　Ｌｅｕ　Ｌｅｕ　Ａｓｎ　Ｓｅｒ　Ａｒｇ　Ｔｈｒ　Ａｒｇ　Ｌｙｓ　Ａｓｎ　Ｔｙｒ　Ｌｅｕ　Ａｌａで表されるアミノ酸配列（配列番号４）を含むＣＤＲ、
（ｅ１）Ｔｒｐ　Ａｌａ　Ｓｅｒ　Ｔｈｅ　Ａｒｇ　Ｇｌｕ　Ｓｅｒで表されるアミノ酸配列（配列番号５）を含むＣＤＲ、及び
（ｆ１）Ｇｌｎ　Ｇｌｎ　Ｓｅｒ　Ｔｙｒ　Ｈｉｓ　Ｌｅｕ　Ｐｒｏ　Ｔｈｒで表されるアミノ酸配列（配列番号６）を含むＣＤＲ
を含むか、あるいは
（ｉｉ）上記（ａ１）~（ｆ１）のＣＤＲを含み（但し、上記（ａ１）~（ｆ１）から選択される１以上（例、１、２、３、４、５又は６）のＣＤＲの各アミノ酸配列において、１又は２アミノ酸残基が置換及び／又は欠失及び／又は付加及び／又は挿入されている）、かつ活性型ＴＧＦ−β１に結合するが、潜在型ＴＧＦ−β１には結合しない抗体である。
　抗体の結合特性は、上記の種々の結合アッセイにより決定することができる。 The antibody (1) of the present invention has (i) the following (a1) to (f1) complementarity determining regions (CDR):
(A1) CDR comprising the amino acid sequence (SEQ ID NO: 1) represented by Gly Tyr Thr Phe Ser Asn Tyr
(B1) CDR comprising the amino acid sequence (Sequence number 2) represented by Tyr Pro Gly Asn Ser Asp,
(C1) a CDR comprising the amino acid sequence (SEQ ID NO: 3) represented by Tyr Ser Asn Tyr Glu Ala Gly Ala Met Asp Tyr;
(D1) a CDR comprising the amino acid sequence (SEQ ID NO: 4) represented by Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Arg Lys Asn Tyr Leu Ala,
(E1) CDR comprising the amino acid sequence (SEQ ID NO: 5) represented by Trp Ala Ser The Arg Glu Ser, and (f1) the amino acid sequence represented by Gln Gln Ser Tyr His Leu Pro Thr (SEQ ID NO: 6) CDR
Or (ii) one or more selected from (a1) to (f1) (eg, 1, 2, 3, 4, 5 or In each amino acid sequence of the CDR of 6), 1 or 2 amino acid residues are substituted and / or deleted and / or added and / or inserted) and active TGF-.beta.1, but latent TGF -It is an antibody which does not bind to β1.
The binding properties of antibodies can be determined by the various binding assays described above.
　好ましくは、本発明の抗体（１）は、
（ｉ）上記（ａ１）~（ｃ１）のＣＤＲを含む重鎖可変領域と、上記（ｄ１）~（ｆ１）のＣＤＲを含む軽鎖可変領域とを含む抗体、あるいは
（ｉｉ）上記（ｉ）の重鎖及び軽鎖可変領域を含み（但し、上記（ａ１）~（ｆ１）から選択される１以上（例、１、２、３、４、５又は６）のＣＤＲの各アミノ酸配列において、１又は２アミノ酸残基が置換及び／又は欠失及び／又は付加及び／又は挿入されている）、かつ活性型ＴＧＦ−β１に結合するが、潜在型ＴＧＦ−β１には結合しない抗体である。 Preferably, the antibody (1) of the present invention is
(I) An antibody comprising a heavy chain variable region comprising the CDRs of (a1) to (c1) and a light chain variable region comprising the CDRs of (d1) to (f1), or (ii) the above (i) In the respective amino acid sequences of one or more (e.g., 1, 2, 3, 4, 5 or 6) CDRs selected from the above (a1) to (f1), including heavy and light chain variable regions of 1 or 2 amino acid residues are substituted and / or deleted and / or added and / or inserted), and an antibody that binds to active TGF-β1 but not latent TGF-β1.
　より好ましくは、上記の抗体において、上記（ａ１）、（ｂ１）及び（ｃ１）のＣＤＲは、重鎖のＮ末端側からこの順で配置されている。即ち、（ａ１）、（ｂ１）及び（ｃ１）のＣＤＲは、それぞれ重鎖のＣＤＲ１、ＣＤＲ２及びＣＤＲ３に相当する。同様に、上記（ｄ１）、（ｅ１）及び（ｆ１）のＣＤＲは、軽鎖のＮ末端側からこの順で配置されている。即ち、（ｄ１）、（ｅ１）及び（ｆ１）のＣＤＲは、それぞれ軽鎖のＣＤＲ１、ＣＤＲ２及びＣＤＲ３に相当する。 More preferably, in the above-mentioned antibody, the CDRs of (a1), (b1) and (c1) above are arranged in this order from the N-terminal side of the heavy chain. That is, the CDRs of (a1), (b1) and (c1) correspond to CDR1, CDR2 and CDR3 of the heavy chain, respectively. Similarly, the CDRs of (d1), (e1) and (f1) above are arranged in this order from the N-terminal side of the light chain. That is, the CDRs of (d1), (e1) and (f1) correspond to CDR1, CDR2 and CDR3 of the light chain, respectively.
　いっそう好ましくは、本発明の抗体（１）は、
（ｉ）配列番号１３で表されるアミノ酸配列を含む重鎖可変領域と、配列番号１４で表されるアミノ酸配列を含む軽鎖可変領域とを含む抗体、あるいは
（ｉｉ）上記（ｉ）の重鎖及び軽鎖可変領域を含み（但し、配列番号１３及び１４のいずれか一方もしくは両方において、１個以上、好ましくは１~２０個、より好ましくは１~１０個、さらに好ましくは１~数個（例、１、２、３、４又は５個）のアミノ酸残基が置換及び／又は欠失及び／又は付加及び／又は挿入されている）、かつ活性型ＴＧＦ−β１に結合するが、潜在型ＴＧＦ−β１には結合しない抗体である。
　配列番号１４で表されるアミノ酸配列において、１１番目の残基がＬｅｕの場合、５０番目の残基は好ましくはＰｒｏであり、１１番目の残基がＡｒｇの場合、５０番目の残基は好ましくはＬｅｕである。 More preferably, the antibody (1) of the present invention is
(I) an antibody comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14; or (ii) the weight of (i) above And 1 or more, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to several, in any one or both of SEQ ID NOs: 13 and 14 (Eg, 1, 2, 3, 4 or 5) amino acid residues are substituted and / or deleted and / or added and / or inserted, and bind to active TGF-β1, Antibodies that do not bind to TGF-.beta.1.
In the amino acid sequence represented by SEQ ID NO: 14, when the 11th residue is Leu, the 50th residue is preferably Pro, and when the 11th residue is Arg, the 50th residue is preferably Is Leu.
　別の実施態様においては、本発明の抗体（１）は、上記のいずれかの抗活性型ＴＧＦ−β１抗体と競合的に活性型ＴＧＦ−β１に結合し、かつ潜在型ＴＧＦ−β１には結合しない抗体である。抗体の競合的結合は、上記の競合アッセイにより測定することができる。 In another embodiment, the antibody (1) of the present invention binds to active TGF-β1 competitively with any of the above-mentioned anti-active TGF-β1 antibodies and binds to latent TGF-β1 Not an antibody. Competitive binding of antibodies can be measured by the above-described competition assay.
　本発明の抗体（２）は、（ｉ）以下の（ａ２）~（ｆ２）の相補性決定領域（ＣＤＲ）：
（ａ２）Ｇｌｙ　Ｔｙｒ　Ｔｈｒ　Ｐｈｅ　Ｔｈｒ　Ａｓｐ　Ｔｙｒで表されるアミノ酸配列（配列番号７）を含むＣＤＲ、
（ｂ２）Ｉｌｅ　Ｐｒｏ　Ａｓｎ　Ｓｅｒ　Ｇｌｙ　Ｇｌｙで表されるアミノ酸配列（配列番号８）を含むＣＤＲ、
（ｃ２）Ｇｌｕ　Ａｌａ　Ｍｅｔ　Ａｓｐ　Ｔｙｒで表されるアミノ酸配列（配列番号９）を含むＣＤＲ、
（ｄ２）Ａｒｇ　Ａｌａ　Ｓｅｒ　Ｇｌｎ　Ｓｅｒ　Ｉｌｅ　Ａｒｇ　Ａｓｎ　Ｌｙｓ　Ｌｅｕ　Ｈｉｓで表されるアミノ酸配列（配列番号１０）を含むＣＤＲ、
（ｅ２）Ｔｙｒ　Ａｌａ　Ｓｅｒ　Ｇｌｎ　Ｓｅｒ　Ｉｌｅ　Ｓｅｒで表されるアミノ酸配列（配列番号１１）を含むＣＤＲ、及び
（ｆ２）Ｌｅｕ　Ｇｌｎ　Ｓｅｒ　Ａｓｎ　Ｓｅｒ　Ｔｒｐ　Ｐｒｏ　Ｌｅｕ　Ｔｈｒで表されるアミノ酸配列（配列番号１２）を含むＣＤＲ
を含むか、あるいは
（ｉｉ）上記（ａ２）~（ｆ２）のＣＤＲを含み（但し、上記（ａ２）~（ｆ２）から選択される１以上（例、１、２、３、４、５又は６）のＣＤＲの各アミノ酸配列において、１又は２アミノ酸残基が置換及び／又は欠失及び／又は付加及び／又は挿入されている）、かつ潜在型ＴＧＦ−β１又はＬＡＰに結合するが、活性型ＴＧＦ−β１には結合しない抗体である。
　抗体の結合特性は、上記の種々の結合アッセイにより決定することができる。 The antibody (2) of the present invention has (i) the following (a2) to (f2) complementarity determining regions (CDR):
(A2) CDR comprising the amino acid sequence (SEQ ID NO: 7) represented by Gly Tyr Thr Phe Thr Asp Tyr
(B2) a CDR comprising the amino acid sequence (SEQ ID NO: 8) represented by Ile Pro Asn Ser Gly Gly,
(C2) a CDR comprising the amino acid sequence (SEQ ID NO: 9) represented by Glu Ala Met Asp Tyr,
(D2) a CDR comprising the amino acid sequence (SEQ ID NO: 10) represented by Arg Ala Ser Gln Ser Ile Arg Asn Lys Leu His,
(E2) a CDR comprising the amino acid sequence (SEQ ID NO: 11) represented by Tyr Ala Ser Gln Ser Ile Ser; and (f 2) the amino acid sequence represented by Leu Gln Ser Asn Ser Trp Pro Leu Thr (SEQ ID NO 12) CDRs included
Or (ii) one or more selected from the above (a2) to (f2) (eg, 1, 2, 3, 4, 5 or In each amino acid sequence of the CDR of 6), one or two amino acid residues are substituted and / or deleted and / or added and / or inserted) and latent TGF-β1 or LAP, but the activity is active Antibodies that do not bind to TGF-.beta.1.
The binding properties of antibodies can be determined by the various binding assays described above.
　好ましくは、本発明の抗体（２）は、
（ｉ）上記（ａ２）~（ｃ２）のＣＤＲを含む重鎖可変領域と、上記（ｄ２）~（ｆ２）のＣＤＲを含む軽鎖可変領域とを含む抗体、あるいは
（ｉｉ）上記（ｉ）の重鎖及び軽鎖可変領域を含み（但し、上記（ａ２）~（ｆ２）から選択される１以上（例、１、２、３、４、５又は６）のＣＤＲの各アミノ酸配列において、１又は２アミノ酸残基が置換及び／又は欠失及び／又は付加及び／又は挿入されている）、かつ潜在型ＴＧＦ−β１又はＬＡＰに結合するが、活性型ＴＧＦ−β１には結合しない抗体である。 Preferably, the antibody (2) of the present invention is
(I) An antibody comprising a heavy chain variable region containing the CDRs of (a2) to (c2) above and a light chain variable region containing the CDRs of (d2) to (f2) above, or (ii) the above (i) In the respective amino acid sequences of one or more (for example, 1, 2, 3, 4, 5 or 6) CDRs selected from the above (a2) to (f2), including the heavy chain and light chain variable regions of An antibody which binds to latent TGF-β1 or LAP but does not bind to active TGF-β1 in which 1 or 2 amino acid residues are substituted and / or deleted and / or added and / or inserted) is there.
　より好ましくは、上記の抗体において、上記（ａ２）、（ｂ２）及び（ｃ２）のＣＤＲは、重鎖のＮ末端側からこの順で配置されている。即ち、（ａ２）、（ｂ２）及び（ｃ２）のＣＤＲは、それぞれ重鎖のＣＤＲ１、ＣＤＲ２及びＣＤＲ３に相当する。同様に、上記（ｄ２）、（ｅ２）及び（ｆ２）のＣＤＲは、軽鎖のＮ末端側からこの順で配置されている。即ち、（ｄ２）、（ｅ２）及び（ｆ２）のＣＤＲは、それぞれ軽鎖のＣＤＲ１、ＣＤＲ２及びＣＤＲ３に相当する。 More preferably, in the above-mentioned antibody, the CDRs of (a2), (b2) and (c2) above are arranged in this order from the N-terminal side of the heavy chain. That is, the CDRs of (a2), (b2) and (c2) correspond to CDR1, CDR2 and CDR3 of the heavy chain, respectively. Similarly, the CDRs of (d2), (e2) and (f2) above are arranged in this order from the N-terminal side of the light chain. That is, the CDRs of (d2), (e2) and (f2) correspond to CDR1, CDR2 and CDR3 of the light chain, respectively.
　いっそう好ましくは、本発明の抗体（２）は、
（ｉ）配列番号１５で表されるアミノ酸配列を含む重鎖可変領域と、配列番号１６で表されるアミノ酸配列を含む軽鎖可変領域とを含む抗体、あるいは
（ｉｉ）上記（ｉ）の重鎖及び軽鎖可変領域を含み（但し、配列番号１５及び１６のいずれか一方もしくは両方において、１個以上、好ましくは１~２０個、より好ましくは１~１０個、さらに好ましくは１~数個（例、１、２、３、４又は５個）のアミノ酸残基が置換及び／又は欠失及び／又は付加及び／又は挿入されている）、かつ潜在型ＴＧＦ−β１又はＬＡＰに結合するが、活性型ＴＧＦ−β１には結合しない抗体である。 More preferably, the antibody (2) of the present invention is
(I) an antibody comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16; or (ii) the weight of (i) above And 1 or more, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to several, in any one or both of SEQ ID NOs: 15 and 16. (Eg, 1, 2, 3, 4 or 5) amino acid residues are substituted and / or deleted and / or added and / or inserted, and bind to latent TGF-β1 or LAP, , An antibody that does not bind to active TGF-β1.
　別の実施態様においては、本発明の抗体（２）は、上記のいずれかの抗潜在型ＴＧＦ−β１／ＬＡＰ抗体と競合的に潜在型ＴＧＦ−β１又はＬＡＰに結合し、かつ活性型ＴＧＦ−β１には結合しない抗体である。抗体の競合的結合は、上記の競合アッセイにより測定することができる。 In another embodiment, the antibody (2) of the present invention binds to latent TGF-β1 or LAP in competition with any of the above-described anti-latent TGF-β1 / LAP antibodies, and activates active TGF- It is an antibody which does not bind to β1. Competitive binding of antibodies can be measured by the above-described competition assay.
　抗体のアイソタイプは特に限定されないが、好ましくはＩｇＧ、ＩｇＭまたはＩｇＡ、特に好ましくはＩｇＧが挙げられる。
　本発明の抗体は、抗原決定基（エピトープ）を特異的に認識し、結合するための相補性決定領域（ＣＤＲ）を少なくとも有するものであれば、分子の形態に特に制限はなく、完全抗体分子の他、例えばＦａｂ、Ｆａｂ’、Ｆ（ａｂ’）２等のフラグメント、ｓｃＦｖ、ｓｃＦｖ−Ｆｃ、ミニボディー、ダイアボディー等の遺伝子工学的に作製されたコンジュゲート分子、あるいはポリエチレングリコール（ＰＥＧ）等のタンパク質安定化作用を有する分子などで修飾されたそれらの誘導体などであってもよい。 The isotype of the antibody is not particularly limited, but preferably it includes IgG, IgM or IgA, particularly preferably IgG.
The antibody of the present invention is not particularly limited as to the form of the molecule as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding an antigenic determinant (epitope), and a complete antibody molecule In addition, for example, fragments such as Fab, Fab ', F (ab') 2 etc., genetically engineered conjugate molecules such as scFv, scFv-Fc, minibody, diabody etc, or polyethylene glycol (PEG) etc. Or a derivative thereof modified with a molecule having the protein stabilizing action of
（ＩＩ−２）本発明の抗体の作製
　本発明の抗体は自体公知の抗体製造法によって作製することができる。以下に、本発明の抗体作製のための免疫原の調製方法、並びに該抗体の製造方法について説明する。 (II-2) Production of Antibody of the Present Invention The antibody of the present invention can be produced by an antibody production method known per se. Hereinafter, a method of preparing an immunogen for producing an antibody of the present invention, and a method of producing the antibody will be described.
（１）免疫原の調製
　本発明の抗体を調製するために使用される抗原としては、潜在型もしくは成熟ＴＧＦ−β１全体又はＬＡＰポリペプチド全体、あるいはその部分ペプチド、あるいはそれと同一の抗原決定基を１種以上有する（合成）ペプチドなどを使用することができる。一実施態様において、６~１５アミノ酸からなる成熟ＴＧＦ−β１又はＬＡＰポリペプチドの部分ペプチドが免疫原として使用される。より好ましくは、成熟ＴＧＦ−β１及びＬＡＰの各領域内の進化的に保存されたユニークなアミノ酸配列を、免疫原として用いることができる。そのような進化的に保存されたユニークなアミノ酸配列は、商業的に利用可能なエピトープ予測ソフトウェアを用いて、ｉｎ　ｓｉｌｉｃｏで予測することができる。 (1) Preparation of Immunogen The antigen used to prepare the antibody of the present invention may be whole latent or mature TGF-β1 or whole LAP polypeptide, or a partial peptide thereof, or an antigenic determinant identical thereto. It is possible to use (synthetic) peptides having one or more kinds. In one embodiment, a partial peptide of mature TGF-β1 or LAP polypeptide consisting of 6 to 15 amino acids is used as an immunogen. More preferably, evolutionarily conserved unique amino acid sequences within the regions of mature TGF-β1 and LAP can be used as immunogens. Such evolutionarily conserved unique amino acid sequences can be predicted in silico using commercially available epitope prediction software.
　潜在型もしくは成熟ＴＧＦ−β１全体又はＬＡＰポリペプチド全体、あるいはその部分ペプチドは、例えば、（ａ）ヒトの組織又は細胞から公知の方法あるいはそれに準ずる方法を用いて調製、（ｂ）ペプチド・シンセサイザー等を使用する公知のペプチド合成法で化学的に合成、（ｃ）潜在型もしくは成熟ＴＧＦ−β１全体又はＬＡＰポリペプチド全体、あるいはその部分ペプチドをコードするＤＮＡを含む形質転換体を培養、あるいは（ｄ）潜在型もしくは成熟ＴＧＦ−β１全体又はＬＡＰポリペプチド全体、あるいはその部分ペプチドをコードする核酸を鋳型として、無細胞転写／翻訳系を用いて生化学的に合成することによって調製される。 The whole latent or mature TGF-β1 or the whole LAP polypeptide, or a partial peptide thereof is prepared, for example, from (a) human tissues or cells using a known method or a method analogous thereto, (b) a peptide synthesizer, etc. (C) culturing a transformant containing a DNA encoding a latent or mature TGF-β1 whole or a whole LAP polypeptide, or a partial peptide thereof; 2.) The whole latent or mature TGF-β1 or the whole LAP polypeptide, or a nucleic acid encoding the partial peptide thereof, is prepared by biochemical synthesis using a cell-free transcription / translation system as a template.
（２）モノクローナル抗体の作製
（ａ）モノクローナル抗体産生細胞の作製
　上記のようにして調製された免疫原は、温血動物に対して、例えば腹腔内注入、静脈注入、皮下注射、皮内注射などの投与方法によって、抗体産生が可能な部位にそれ自体単独であるいは担体、希釈剤と共に投与される。投与に際して抗体産生能を高めるため、完全フロイントアジュバントや不完全フロイントアジュバントを投与してもよい。投与は、通常１~６週毎に１回ずつ、計２~１０回程度行われる。温血動物としては、例えばマウス、ラット、ウサギ、ヤギ、サル、イヌ、モルモット、ヒッジ、ロバ、ニワトリ等が用いられるが、マウス、ラット及びウサギが好ましい。 (2) Preparation of Monoclonal Antibody (a) Preparation of Monoclonal Antibody-Producing Cell The immunogen prepared as described above is used for warm-blooded animals, for example, intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, etc. Depending on the method of administration, it is administered to the site where antibody production is possible either alone or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance antibody production ability upon administration. The administration is usually performed once every 1 to 6 weeks, for a total of about 2 to 10 times. As a warm-blooded animal, for example, a mouse, a rat, a rabbit, a goat, a monkey, a dog, a guinea pig, a hedge, a donkey, a chicken and the like are used, but a mouse, a rat and a rabbit are preferable.
　あるいは、免疫原を体外免疫法に供することもできる。体外免疫法に用いられる動物細胞としては、ヒトおよび上記した温血動物（好ましくはマウス、ラット）の末梢血、脾臓、リンパ節などから単離されるリンパ球、好ましくはＢリンパ球等が挙げられる。例えば、マウスやラット細胞の場合、４~１２週齢程度の動物から脾臓を摘出・脾細胞を分離し、適当な培地（例：ダルベッコ改変イーグル培地（ＤＭＥＭ）、ＲＰＭＩ１６４０培地、ハムＦ１２培地等）で洗浄した後、抗原を含む胎仔ウシ血清（ＦＣＳ；５~２０％程度）添加培地に浮遊させて４~１０日間程度ＣＯ２インキュベーターなどを用いて培養する。抗原濃度としては、例えば０．０５~５μｇが挙げられるがこれに限定されない。同一系統の動物（１~２週齢程度が好ましい）の胸腺細胞培養上清を常法に従って調製し、培地に添加することが好ましい。 Alternatively, the immunogen can be subjected to extracorporeal immunization. Examples of animal cells used for extracorporeal immunization include lymphocytes isolated from human and the above-mentioned warm-blooded animals (preferably mice and rats), peripheral blood, spleen, lymph nodes and the like, preferably B lymphocytes and the like. . For example, in the case of mouse or rat cells, spleen is removed from an animal of about 4 to 12 weeks of age and spleen cells are separated and an appropriate medium (eg, Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, Ham's F12 medium, etc.) After washing with the medium, the cells are suspended in a medium supplemented with fetal bovine serum (FCS; about 5 to 20%) containing an antigen, and cultured using a CO 2 incubator or the like for about 4 to 10 days. The antigen concentration includes, for example, 0.05 to 5 μg, but is not limited thereto. It is preferable to prepare thymocyte culture supernatants of animals of the same strain (preferably about 1 to 2 weeks old) according to a conventional method and to add to the medium.
　ヒト細胞の体外免疫では、胸腺細胞培養上清を得ることは困難なので、ＩＬ−２、ＩＬ−４、ＩＬ−５、ＩＬ−６等数種のサイトカインおよび必要に応じてアジュバント物質（例：ムラミルジペプチド等）を抗原とともに培地に添加して免疫感作を行うことが好ましい。 Since it is difficult to obtain thymocyte culture supernatant by external immunization of human cells, several cytokines such as IL-2, IL-4, IL-5, and IL-6 and adjuvant substances as needed (eg, unevenness) Preferably, the immunization is carried out by adding a mildipeptide or the like) to the medium together with the antigen.
　モノクローナル抗体の作製に際しては、抗原を免疫された温血動物（例：マウス、ラット）もしくは動物細胞（例：ヒト、マウス、ラット）から抗体価の上昇が認められた個体もしくは細胞集団を選択し、最終免疫の２~５日後に脾臓またはリンパ節を採取もしくは体外免疫後４~１０日間培養した後に細胞を回収して抗体産生細胞を単離し、これと骨髄腫細胞とを融合させることにより抗体産生ハイブリドーマを調製することができる。血清中の抗体価の測定は、例えば標識化抗原と抗血清とを反応させた後、抗体に結合した標識剤の活性を測定することにより行うことができる。 In the preparation of monoclonal antibodies, individuals or cell populations with elevated antibody titers are selected from warm-blooded animals (eg, mice, rats) or animal cells (eg, humans, mice, rats) immunized with an antigen. After harvesting the spleen or lymph node 2 to 5 days after the final immunization, or culturing it for 4 to 10 days after extracorporeal immunization, recover the cells, isolate the antibody-producing cells, and fuse this with myeloma cells. Production hybridomas can be prepared. The antibody titer in serum can be measured, for example, by reacting a labeled antigen with an antiserum and then measuring the activity of the labeling agent bound to the antibody.
　骨髄腫細胞は多量の抗体を分泌するハイブリドーマを産生し得るものであれば特に制限はないが、自身は抗体を産生もしくは分泌しないものが好ましく、また、細胞融合効率が高いものがより好ましい。また、ハイブリドーマの選択を容易にするために、ＨＡＴ（ヒポキサンチン、アミノプテリン、チミジン）感受性の株を用いることが好ましい。例えばマウス骨髄腫細胞としてはＮＳ−１、Ｐ３Ｕ１、ＳＰ２／０、ＡＰ−１等が、ラット骨髄腫細胞としてはＲ２１０．ＲＣＹ３、Ｙ３−Ａｇ　１．２．３等が、ヒト骨髄腫細胞としてはＳＫＯ−００７、ＧＭ１５００−６ＴＧ−２、ＬＩＣＲ−ＬＯＮ−ＨＭｙ２、ＵＣ７２９−６等が挙げられる。 The myeloma cells are not particularly limited as long as they can produce hybridomas that secrete a large amount of antibodies, but those that do not themselves produce or secrete antibodies are preferable, and those with high cell fusion efficiency are more preferable. Also, in order to facilitate selection of hybridomas, it is preferable to use a HAT (hypoxanthine, aminopterin, thymidine) sensitive strain. For example, mouse myeloma cells include NS-1, P3U1, SP2 / 0, AP-1 and the like, and rat myeloma cells include R210. RCY3, Y3-Ag 1.2.3 and the like, and human myeloma cells include SKO-007, GM1500-6TG-2, LICR-LON-HMy2, UC729-6 and the like.
　融合操作は既知の方法、例えばケーラーとミルスタインの方法［ネイチャー（Ｎａｔｕｒｅ）、２５６巻、４９５頁（１９７５年）］に従って実施することができる。融合促進剤としては、ポリエチレングリコール（ＰＥＧ）やセンダイウィルスなどが挙げられるが、好ましくはＰＥＧなどが用いられる。ＰＥＧの分子量は特に制限はないが、低毒性で且つ粘性が比較的低いＰＥＧ１０００~ＰＥＧ６０００が好ましい。ＰＥＧ濃度としては例えば１０~８０％程度、好ましくは３０~５０％程度が例示される。ＰＥＧの希釈用溶液としては無血清培地（例：ＲＰＭＩ１６４０）、５~２０％程度の血清を含む完全培地、リン酸緩衝生理食塩水（ＰＢＳ）、トリス緩衝液等の各種緩衝液を用いることができる。所望によりＤＭＳＯ（例：１０~２０％程度）を添加することもできる。融合液のｐＨとしては、例えば４~１０程度、好ましくは６~８程度が挙げられる。 The fusion operation can be carried out according to known methods, for example, the method of Koehler and Milstein (Nature, vol. 256, p. 495 (1975)). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and preferably PEG is used. The molecular weight of PEG is not particularly limited, but low toxicity and relatively low viscosity PEG 1000 to PEG 6000 are preferable. The PEG concentration is, for example, about 10 to 80%, preferably about 30 to 50%. As a solution for dilution of PEG, various buffers such as serum-free medium (eg RPMI 1640), complete medium containing about 5 to 20% serum, phosphate buffered saline (PBS), Tris buffer etc. may be used it can. If desired, DMSO (eg, about 10 to 20%) can also be added. The pH of the fusion solution is, for example, about 4 to 10, preferably about 6 to 8.
　抗体産生細胞（脾細胞）数と骨髄腫細胞数との好ましい比率は、通常１：１~２０：１程度であり、通常２０~４０℃、好ましくは３０~３７℃で通常１~１０分間インキュベートすることにより効率よく細胞融合を実施できる。 The preferred ratio of the number of antibody-producing cells (splenocytes) to the number of myeloma cells is usually about 1: 1 to 20: 1, usually 20 to 40 ° C., preferably 30 to 37 ° C. and usually 1 to 10 minutes of incubation Cell fusion can be performed efficiently.
　抗体産生細胞株はまた、リンパ球をトランスフォームし得るウイルスに抗体産生細胞を感染させて該細胞を不死化することによっても得ることができる。そのようなウイルスとしては、例えばエプスタイン−バー（ＥＢ）ウイルス等が挙げられる。大多数の人は伝染性単核球症の無症状感染としてこのウイルスに感染した経験があるので免疫を有しているが、通常のＥＢウイルスを用いた場合にはウイルス粒子も産生されるので、適切な精製を行うべきである。ウイルス混入の可能性のないＥＢシステムとして、Ｂリンパ球を不死化する能力を保持するがウイルス粒子の複製能力を欠損した組換えＥＢウイルス（例えば、潜伏感染状態から溶解感染状態への移行のスイッチ遺伝子における欠損など）を用いることもまた好ましい。 Antibody-producing cell lines can also be obtained by infecting antibody-producing cells with a virus capable of transforming lymphocytes to immortalize the cells. As such a virus, for example, Epstein-Barr (EB) virus and the like can be mentioned. The majority of people are immune because they have been infected with this virus as a subclinical infection with infectious mononucleosis, but they also produce virus particles when using the regular EB virus. And should be properly purified. A recombinant EB virus that retains the ability to immortalize B lymphocytes but lacks the ability of viral particles to replicate as an EB system without the possibility of viral contamination (eg, switches the transition from latent to lytic state) It is also preferred to use such as a deletion in the gene.
　マーモセット由来のＢ９５−８細胞はＥＢウイルスを分泌しているので、その培養上清を用いれば容易にＢリンパ球をトランスフォームすることができる。この細胞を例えば血清及びペニシリン／ストレプトマイシン（Ｐ／Ｓ）添加培地（例：ＲＰＭＩ１６４０）もしくは細胞増殖因子を添加した無血清培地で培養した後、濾過もしくは遠心分離等により培養上清を分離し、これに抗体産生Ｂリンパ球を適当な濃度（例：約１０７細胞／ｍＬ）で浮遊させて、通常２０~４０℃、好ましくは３０~３７℃で通常０．５~２時間程度インキュベートすることにより抗体産生Ｂ細胞株を得ることができる。ヒトの抗体産生細胞が混合リンパ球として提供される場合、大部分の人はＥＢウイルス感染細胞に対して傷害性を示すＴリンパ球を有しているので、トランスフォーメーション頻度を高めるためには、例えばヒツジ赤血球等とＥロゼットを形成させることによってＴリンパ球を予め除去しておくことが好ましい。また、可溶性抗原を結合したヒツジ赤血球を抗体産生Ｂリンパ球と混合し、パーコール等の密度勾配を用いてロゼットを分離することにより標的抗原に特異的なリンパ球を選別することができる。さらに、大過剰の抗原を添加することにより抗原特異的なＢリンパ球はキャップされて表面にＩｇＧを提示しなくなるので、抗ＩｇＧ抗体を結合したヒツジ赤血球と混合すると抗原非特異的なＢリンパ球のみがロゼットを形成する。従って、この混合物からパーコール等の密度勾配を用いてロゼット非形成層を採取することにより、抗原特異的Ｂリンパ球を選別することができる。 Since B95-8 cells derived from marmoset secrete EB virus, B lymphocytes can be easily transformed by using the culture supernatant. The cells are cultured in, for example, serum and a medium supplemented with serum and penicillin / streptomycin (P / S) (eg RPMI 1640) or serum free medium to which cell growth factor has been added, and the culture supernatant is separated by filtration or centrifugation. The antibody-producing B lymphocytes are suspended at a suitable concentration (eg, about 10 7 cells / mL) and incubated usually at about 20 to 40 ° C., preferably 30 to 37 ° C. for about 0.5 to 2 hours. Antibody-producing B cell lines can be obtained. When human antibody-producing cells are provided as mixed lymphocytes, most people have T lymphocytes that show toxicity to EB virus-infected cells, so to increase transformation frequency, For example, T lymphocytes are preferably removed in advance by forming E rosettes with sheep red blood cells and the like. Alternatively, lymphocytes specific to a target antigen can be selected by mixing soluble erythrocyte-bound sheep red blood cells with antibody-producing B lymphocytes and separating rosettes using a density gradient such as Percoll. Furthermore, the addition of a large excess of antigen results in capping of antigen-specific B lymphocytes without presenting IgG on the surface, so that antigen non-specific B lymphocytes when mixed with sheep red blood cells conjugated with anti-IgG antibodies Only form a rosette. Therefore, antigen-specific B lymphocytes can be sorted from this mixture by collecting a rosette non-forming layer using a density gradient such as Percoll.
　トランスフォーメーションによって無限増殖能を獲得したヒト抗体分泌細胞は、抗体分泌能を安定に持続させるためにマウスもしくはヒトの骨髄腫細胞と戻し融合させることができる。骨髄腫細胞としては上記と同様のものが用いられ得る。 Human antibody-secreting cells that have acquired infinite proliferation ability by transformation can be back-fused with mouse or human myeloma cells in order to stably sustain antibody secreting ability. The same myeloma cells as those described above can be used.
　ハイブリドーマのスクリーニング、育種は通常ＨＡＴ（ヒポキサンチン、アミノプテリン、チミジン）を添加して、５~２０％ＦＣＳを含む動物細胞用培地（例：ＲＰＭＩ１６４０）もしくは細胞増殖因子を添加した無血清培地で行われる。ヒポキサンチン、アミノプテリンおよびチミジンの濃度としては、例えばそれぞれ約０．１ｍＭ、約０．４μＭおよび約０．０１６ｍＭ等が挙げられる。ヒト−マウスハイブリドーマの選択にはウワバイン耐性を用いることができる。ヒト細胞株はマウス細胞株に比べてウワバインに対する感受性が高いので、１０−７~１０−３Ｍ程度で培地に添加することにより未融合のヒト細胞を排除することができる。 Screening and breeding of hybridomas are usually performed by adding HAT (hypoxanthine, aminopterin, thymidine) and using a medium for animal cells (eg RPMI 1640) containing 5-20% FCS or a serum-free medium supplemented with cell growth factors. It will be. The concentrations of hypoxanthine, aminopterin and thymidine include, for example, about 0.1 mM, about 0.4 μM and about 0.016 mM, respectively. For selection of human-mouse hybridomas, ouabain resistance can be used. Since human cell lines are more sensitive to ouabain than mouse cell lines, unfused human cells can be excluded by adding to the medium at about 10 -7 to 10 -3 M.
　ハイブリドーマの選択にはフィーダー細胞やある種の細胞培養上清を用いることが好ましい。フィーダー細胞としては、ハイブリドーマの出現を助けて自身は死滅するように生存期間が限られた異系の細胞種、ハイブリドーマの出現に有用な増殖因子を大量に産生し得る細胞を放射線照射等して増殖力を低減させたもの等が用いられる。例えば、マウスのフィーダー細胞としては、脾細胞、マクロファージ、血液、胸腺細胞等が、ヒトのフィーダー細胞としては、末梢血単核細胞等が挙げられる。細胞培養上清としては、例えば上記の各種細胞の初代培養上清や種々の株化細胞の培養上清が挙げられる。 It is preferable to use feeder cells and certain cell culture supernatants for selection of hybridomas. As feeder cells, allogeneic cell types with a limited survival time to help the emergence of hybridomas and die themselves, irradiation with cells that can produce large amounts of growth factors useful for emergence of hybridomas, etc. Those having reduced proliferation ability are used. For example, mouse feeder cells include splenocytes, macrophages, blood, thymocytes and the like, and human feeder cells include peripheral blood mononuclear cells and the like. Examples of the cell culture supernatant include primary culture supernatants of the various cells described above and culture supernatants of various established cell lines.
　また、ハイブリドーマは、抗原を蛍光標識して融合細胞と反応させた後、蛍光活性化セルソータ（ＦＡＣＳ）を用いて抗原と結合する細胞を分離することによっても選択することができる。この場合、標的抗原に対する抗体を産生するハイブリドーマを直接選択することができるので、クローニングの労力を大いに軽減することが可能である。 Alternatively, hybridomas can also be selected by fluorescently labeling the antigen and reacting it with the fused cells, and then separating out cells that bind to the antigen using a fluorescence activated cell sorter (FACS). In this case, the cloning effort can be greatly reduced since hybridomas producing antibodies against the target antigen can be directly selected.
　標的抗原に対するモノクローナル抗体を産生するハイブリドーマのクローニングには種々の方法が使用できる。 Various methods can be used to clone a hybridoma producing a monoclonal antibody against a target antigen.
　アミノプテリンは多くの細胞機能を阻害するので、できるだけ早く培地から除去することが好ましい。マウスやラットの場合、ほとんどの骨髄腫細胞は１０~１４日以内に死滅するので、融合２週間後からはアミノプテリンを除去することができる。但し、ヒトハイブリドーマについては通常融合後４~６週間程度はアミノプテリン添加培地で維持される。ヒポキサンチン、チミジンはアミノプテリン除去後１週間以上後に除去するのが望ましい。即ち、マウス細胞の場合、例えば融合７~１０日後にヒポキサンチンおよびチミジン（ＨＴ）添加完全培地（例：１０％　ＦＣＳ添加ＲＰＭＩ１６４０）の添加または交換を行う。融合後８~１４日程度で目視可能なクローンが出現する。クローンの直径が１ｍｍ程度になれば培養上清中の抗体量の測定が可能となる。 As aminopterin inhibits many cellular functions, it is preferable to remove it from the medium as soon as possible. In the case of mice and rats, most myeloma cells die within 10-14 days, and aminopterin can be removed two weeks after fusion. However, human hybridomas are usually maintained in an aminopterin-containing medium for about 4 to 6 weeks after fusion. It is desirable to remove hypoxanthine and thymidine one week or more after aminopterin removal. That is, in the case of mouse cells, for example, addition or replacement of hypoxanthine and thymidine (HT) -added complete medium (eg, RPMI 1640 supplemented with 10% FCS) is performed 7 to 10 days after fusion. Visually visible clones appear about 8 to 14 days after fusion. When the diameter of the clone is about 1 mm, the amount of antibody in the culture supernatant can be measured.
　抗体量の測定は、例えば標的抗原またはその誘導体あるいはその部分ペプチド（抗原決定基として用いた部分アミノ酸配列を含む）を直接あるいは担体とともに吸着させた固相（例：マイクロプレート）にハイブリドーマ培養上清を添加し、次に放射性物質（例：１２５Ｉ、１３１Ｉ、３Ｈ、１４Ｃ）、酵素（例：β−ガラクトシダーゼ、β−グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素）、蛍光物質（例：フルオレスカミン、フルオレッセンイソチオシアネート）、発光物質（例：ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニン）などで標識した抗免疫グロブリン（ＩｇＧ）抗体（もとの抗体産生細胞が由来する動物と同一種の動物由来のＩｇＧに対する抗体が用いられる）またはプロテインＡを加え、固相に結合した標的抗原（抗原決定基）に対する抗体を検出する方法、抗ＩｇＧ抗体またはプロテインＡを吸着させた固相にハイブリドーマ培養上清を添加し、上記と同様の標識剤で標識した標的抗原またはその誘導体あるいはその部分ペプチドを加え、固相に結合した標的抗原（抗原決定基）に対する抗体を検出する方法などによって行うことができる。 The antibody amount can be measured, for example, by using a hybridoma culture supernatant on a solid phase (eg, microplate) on which a target antigen or a derivative thereof or a partial peptide thereof (including a partial amino acid sequence used as an antigenic determinant) is adsorbed directly or with a carrier. And then radioactive substances (eg 125 I, 131 I, 3 H, 14 C), enzymes (eg β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase), fluorescence Anti-immunoglobulin (IgG) antibodies (eg, from which the original antibody-producing cells are labeled) with substances (eg, fluorescamine, fluorescein isothiocyanate), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin), etc. (Antibodies against IgG from the same kind of animals are used) Method for detecting protein against target antigen (antigenic determinant) bound to solid phase by adding protein A, adding hybridoma culture supernatant to the solid phase adsorbed with anti-IgG antibody or protein A, and labeling as described above It can be carried out by a method of detecting an antibody against a target antigen (antigenic determinant) bound to a solid phase by adding an agent-labeled target antigen or a derivative thereof or a partial peptide thereof.
　クローニング方法としては限界希釈法が通常用いられるが、軟寒天を用いたクローニングやＦＡＣＳを用いたクローニング（上述）も可能である。限界希釈法によるクローニングは、例えば以下の手順で行うことができるがこれに限定されない。 Although a limiting dilution method is usually used as a cloning method, cloning using soft agar and cloning using FACS (described above) are also possible. The cloning by the limiting dilution method can be performed, for example, by the following procedure, but is not limited thereto.
　上記のようにして抗体量を測定して陽性ウェルを選択する。適当なフィーダー細胞を選択して９６ウェルプレートに添加しておく。抗体陽性ウェルから細胞を吸い出し、完全培地（例：１０％　ＦＣＳおよびＰ／Ｓ添加ＲＭＰＩ１６４０）中に３０細胞／ｍＬの密度となるように浮遊させ、フィーダー細胞を添加したウェルプレートに０．１ｍＬ（３細胞／ウェル）加え、残りの細胞懸濁液を１０細胞／ｍＬに希釈して別のウェルに同様にまき（１細胞／ウェル）、さらに残りの細胞懸濁液を３細胞／ｍＬに希釈して別のウェルにまく（０．３細胞／ウェル）。目視可能なクローンが出現するまで２~３週間程度培養し、抗体量を測定・陽性ウェルを選択し、再度クローニングする。ヒト細胞の場合はクローニングが比較的困難なので、１０細胞／ウェルのプレートも調製しておく。通常２回のサブクローニングでモノクローナル抗体産生ハイブリドーマを得ることができるが、その安定性を確認するためにさらに数ヶ月間定期的に再クローニングを行うことが望ましい。 The amount of antibody is measured as described above to select positive wells. Appropriate feeder cells are selected and added to the 96 well plate. Aspirate cells from antibody-positive wells, suspend in complete medium (eg 10% FCS and P / S supplemented RMPI 1640) to a density of 30 cells / mL, and add 0.1 mL to the well plate to which feeder cells have been added. Add 3 cells / well), dilute the remaining cell suspension to 10 cells / mL, and in the same way to another well (1 cell / well), further dilute the remaining cell suspension to 3 cells / mL Then in another well (0.3 cells / well). The cells are cultured for about 2 to 3 weeks until visible clones appear, the amount of antibody is measured, and positive wells are selected and cloned again. Plates of 10 cells / well are also prepared because cloning is relatively difficult in the case of human cells. Although monoclonal antibody-producing hybridomas can usually be obtained by two subclonings, it is desirable to carry out periodical reclonings for several more months to confirm the stability.
（ｂ）ディファレンシャル・スクリーニング
　上記のようにして得られた活性型ＴＧＦ−β１に対するモノクローナル抗体を産生するハイブリドーマは、次いで２次スクリーニングに供される。２次スクリーニングでは、免疫原として使用された活性型ＴＧＦ−β１及び／又はその部分ペプチドだけでなく、潜在型ＴＧＦ−β１もプローブとして使用される。上記のようにして得られた潜在型ＴＧＦ−β１／ＬＡＰに対するモノクローナル抗体を産生するハイブリドーマもまた、次いで２次スクリーニングに供される。２次スクリーニングでは、免疫原として使用された潜在型ＴＧＦ−β１及び／又はＬＡＰの部分ペプチドだけでなく、活性型ＴＧＦ−β１もプローブとして使用される。２次スクリーニングの結果、活性型ＴＧＦ−β１及び／又はその部分ペプチドとは反応するが、潜在型ＴＧＦ−β１とは反応しなかったモノクローナル抗体を産生するハイブリドーマを、本発明の抗活性型ＴＧＦ−β１抗体を産生するハイブリドーマとして選択することができる。同様に、２次スクリーニングの結果、潜在型ＴＧＦ−β１及び／又はＬＡＰの部分ペプチドとは反応するが、活性型ＴＧＦ−β１とは反応しなかったモノクローナル抗体を産生するハイブリドーマを、本発明の抗潜在型ＴＧＦ−β１／ＬＡＰ抗体を産生するハイブリドーマとして選択することができる。 (B) Differential screening The hybridoma producing the monoclonal antibody against active TGF-β1 obtained as described above is then subjected to a secondary screening. In the secondary screening, not only active TGF-β1 used as an immunogen and / or its partial peptide, but also latent TGF-β1 is used as a probe. Hybridomas producing monoclonal antibodies against latent TGF-β1 / LAP obtained as described above are also subjected to secondary screening. In the secondary screening, not only the latent TGF-β1 and / or the partial peptide of LAP used as the immunogen, but also the active TGF-β1 is used as a probe. As a result of the secondary screening, a hybridoma that produces a monoclonal antibody that reacts with active TGF-β1 and / or its partial peptide but does not react with latent TGF-β1 is referred to as anti-active TGF- of the present invention. It can be selected as a hybridoma that produces β1 antibody. Similarly, as a result of the secondary screening, a hybridoma producing a monoclonal antibody which reacts with latent TGF-β1 and / or a partial peptide of LAP but does not react with active TGF-β1 is used as an anti-tumor agent of the present invention. It can be selected as a hybridoma that produces latent TGF-β1 / LAP antibody.
（ｃ）ネガティブ選択
　好ましい一実施態様において、本発明の抗体は、それが認識するエピトープのアミノ酸配列から予測される、ＴＧＦ−β２、ＴＧＦ−β３等を含むいかなるオフターゲットタンパク質とも交差反応しない。従って、上記のようにして得られた活性型ＴＧＦ−β１又は潜在型ＴＧＦ−β１／ＬＡＰのいずれか一方のみを認識するモノクローナル抗体を産生するハイブリドーマをネガティブ選択にかけ、それらがオフターゲットタンパク質と交差反応しないことを確認することができる。 (C) Negative Selection In a preferred embodiment, the antibody of the present invention does not cross react with any off-target proteins, including TGF-β2, TGF-β3, etc. as predicted from the amino acid sequence of the epitope that it recognizes. Therefore, hybridomas producing monoclonal antibodies that recognize only either active TGF-β1 or latent TGF-β1 / LAP obtained as described above are subjected to negative selection, and they cross react with off-target proteins. You can confirm that you do not.
　こうして得られたハイブリドーマはｉｎ　ｖｉｔｒｏ又はｉｎ　ｖｉｖｏで培養するすることができる。ｉｎ　ｖｉｔｒｏでの培養法としては、上記のようにして得られるモノクローナル抗体産生ハイブリドーマを、細胞密度を例えば１０５~１０６細胞／ｍＬ程度に保ちながら、また、ＦＣＳ濃度を徐々に減らしながら、ウェルプレートから徐々にスケールアップしていく方法が挙げられる。ｉｎ　ｖｉｖｏでの培養法としては、例えば、腹腔内にミネラルオイルを注入して形質細胞腫（ＭＯＰＣ）を誘導したマウス（ハイブリドーマの親株と組織適合性のマウス）に、５~１０日後に１０６~１０７細胞程度のハイブリドーマを腹腔内注射し、２~５週間後に麻酔下で腹水を採取する方法が挙げられる。 The hybridoma thus obtained can be cultured in vitro or in vivo. As an in vitro culture method, the monoclonal antibody-producing hybridoma obtained as described above may be used as a well while maintaining the cell density at, for example, about 10 5 to 10 6 cells / mL and gradually reducing the FCS concentration. There is a method of gradually scaling up from the plate. As an in vivo culture method, for example, mice that have been injected with mineral oil into the peritoneal cavity to induce plasmacytoma (MOPC) (10 mice that are histocompatibility with the parent strain of the hybridoma) may be 10 6 days later. There is a method of intraperitoneally injecting a hybridoma of about 10 7 cells and collecting ascites fluid under anesthesia 2 to 5 weeks later.
（ｄ）モノクローナル抗体の精製
　モノクローナル抗体の分離精製は、自体公知の方法、例えば、免疫グロブリンの分離精製法［例：塩析法、アルコール沈殿法、等電点沈殿法、電気泳動法、イオン交換体（例：ＤＥＡＥ、ＱＥＡＥ）による吸脱着法、超遠心法、ゲルろ過法、抗原結合固相あるいはプロテインＡあるいはプロテインＧなどの活性吸着剤により抗体のみを採取し、結合を解離させて抗体を得る特異的精製法など］に従って行うことができる。
　以上のようにして、ハイブリドーマを温血動物の生体内又は生体外で培養し、その体液または培養物から抗体を採取することによって、モノクローナル抗体を製造することができる。 (D) Purification of monoclonal antibody Separation and purification of monoclonal antibody can be carried out by a method known per se, for example, separation and purification of immunoglobulin [eg salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchange The antibody alone is collected by adsorption / desorption method by the body (eg DEAE, QEAE), ultracentrifugation, gel filtration, antigen binding solid phase or active adsorbent such as protein A or protein G, and the binding is dissociated to obtain antibody. Can be performed according to the specific purification method to be obtained.
As described above, a monoclonal antibody can be produced by culturing a hybridoma in or on a warm-blooded animal and collecting the antibody from its body fluid or culture.
　本発明の抗体（１）の例として、後記実施例に記載されるマウス抗活性型ＴＧＦ−β１抗体クローン２Ｈ４及び４Ｄ１０が挙げられる。また、本発明の抗体（２）の例として、後記実施例に記載されるマウス抗潜在型ＴＧＦ−β１抗体クローン２Ｆ１０が挙げられる。アミノ酸配列決定の結果、２Ｈ４及び４Ｄ１０抗体は、配列番号１３で表されるアミノ酸配列からなる重鎖可変領域と、配列番号１４で表されるアミノ酸配列（但し、２Ｈ４抗体の場合、１１番目及び５０番目の残基はそれぞれＬｅｕ及びＰｒｏであり、４Ｄ１０抗体の場合、１１番目及び５０番目の残基はそれぞれＡｒｇ及びＬｅｕである）からなる軽鎖可変領域とを有することが明らかとなった。また、２Ｆ１０抗体は、配列番号１５で表されるアミノ酸配列からなる重鎖可変領域と、配列番号１６で表されるアミノ酸配列からなる軽鎖可変領域とを有することが明らかとなった。 Examples of the antibody (1) of the present invention include mouse anti-active TGF-β1 antibody clones 2H4 and 4D10 described in the Examples below. In addition, as an example of the antibody (2) of the present invention, a mouse anti-latent TGF-β1 antibody clone 2F10 described in the following Examples can be mentioned. As a result of amino acid sequencing, the 2H4 and 4D10 antibodies consist of the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 14 (however, 11th and 50th in the case of 2H4 antibody) The th residue is Leu and Pro, respectively, and in the case of the 4D10 antibody, it has been revealed that the 11th and 50th residues are Arg and Leu, respectively, and a light chain variable region. The 2F10 antibody was also found to have a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16.
（ｅ）組換え抗体の作製
　別の実施態様において、こうして得られた抗活性型ＴＧＦ−β１抗体又は抗潜在型ＴＧＦ−β１／ＬＡＰ抗体の重鎖及び軽鎖をコードするｃＤＮＡを、該抗体を産生するハイブリドーマのｃＤＮＡライブラリーから単離し、常法に従って、目的の宿主細胞で機能的な適当な発現ベクターにクローニングすることができる。次いで、こうして得られた重鎖及び軽鎖発現ベクターを宿主細胞に導入する。有用な宿主細胞としては、動物細胞、例えば上記したマウス骨髄腫細胞の他、チャイニーズハムスター卵巣（ＣＨＯ）細胞、サル由来のＣＯＳ−７細胞、Ｖｅｒｏ細胞、ラット由来のＧＨＳ細胞などが挙げられる。遺伝子導入は動物細胞に適用可能ないかなる方法を用いてもよいが、好ましくはエレクトロポレーション法又はカチオン性脂質を用いた方法などが挙げられる。宿主細胞に適した培地中で一定期間培養後、培養上清を回収して抗体タンパク質を常法により精製することにより、本発明の抗体を単離することができる。あるいは、宿主細胞としてウシ、ヤギ、ニワトリ等のトランスジェニック技術が確立し、且つ家畜（家禽）として大量繁殖のノウハウが蓄積されている動物の生殖系列細胞を用い、常法によってトランスジェニック動物を作製することにより、得られる動物の乳汁もしくは卵から容易に且つ大量に本発明の抗体を得ることもできる。さらに、トウモロコシ、イネ、コムギ、ダイズ、タバコなどのトランスジェニック技術が確立し、且つ主要作物として大量に栽培されている植物細胞を宿主細胞として、プロトプラストへのマイクロインジェクションやエレクトロポレーション、無傷細胞へのパーティクルガン法やＴｉベクター法などを用いてトランスジェニック植物を作製し、得られる種子や葉などから大量に本発明の抗体を得ることも可能である。 (E) Preparation of Recombinant Antibody In another embodiment, the cDNA encoding the heavy chain and light chain of the thus-obtained anti-active TGF-β1 antibody or anti-latent TGF-β1 / LAP antibody is used as the antibody. It can be isolated from a cDNA library of producing hybridomas and cloned into an appropriate expression vector functional in a desired host cell according to a conventional method. The heavy and light chain expression vectors thus obtained are then introduced into host cells. Useful host cells include animal cells, such as mouse myeloma cells as described above, Chinese hamster ovary (CHO) cells, COS-7 cells from monkeys, Vero cells, GHS cells from rats, etc. For gene transfer, any method applicable to animal cells may be used, but preferably, electroporation, a method using a cationic lipid, and the like can be mentioned. After culturing for a fixed period in a medium suitable for host cells, the culture supernatant can be recovered and the antibody protein can be isolated by conventional purification of the antibody protein. Alternatively, transgenic animals are produced by a conventional method using germline cells of animals in which transgenic techniques such as cattle, goats and chickens have been established as host cells, and know-how on mass breeding has been accumulated as livestock (poultry). By doing this, the antibody of the present invention can also be obtained easily and in large amounts from the milk or egg of the animal obtained. In addition, plant cells that have been established as transgenic plants such as maize, rice, wheat, soybean, and tobacco, and are grown in large quantities as major crops, are used as host cells for microinjection and electroporation into protoplasts, and intact cells. It is also possible to produce a transgenic plant using the particle gun method or Ti vector method of the above, and to obtain the antibody of the present invention in large amounts from the resulting seeds and leaves.
（ＩＩ−３）本発明の検査方法
　本発明の検査方法は、がんもしくは線維症患者、又はその疑いのある者由来の検体と、活性型ＴＧＦ−β１に結合するが、潜在型ＴＧＦ−β１には結合しない第１のモノクローナル抗体（本発明の抗体（１））、及び／又は、潜在型ＴＧＦ−β１もしくはＬＡＰに結合するが、活性型ＴＧＦ−β１には結合しない第２のモノクローナル抗体（本発明の抗体（２））とを、接触させ、該検体中の活性型ＴＧＦ−β１及び／又は潜在型ＴＧＦ−β１もしくはＬＡＰを特異的に検出することを特徴とする。 (II-3) The test method of the present invention The test method of the present invention binds to active TGF-β1 with a sample from a cancer or fibrotic patient or a person suspected of it, but latent TGF-β1 A first monoclonal antibody (antibody (1) of the present invention) that does not bind to the antibody, and / or a second monoclonal antibody that binds to latent TGF-β1 or LAP but not to active TGF-β1 ( It is characterized in that it is contacted with the antibody (2) of the present invention to specifically detect active TGF-β1 and / or latent TGF-β1 or LAP in the sample.
　本発明の検査方法の対象となる「がん」は特に制限されず、例えば、上皮細胞由来の癌であり得るが、非上皮性の肉腫や血液がんであってもよい。より具体的には、例えば、頭頸部のがん（例えば、上顎がん、咽頭がん、喉頭がん、舌がん、甲状腺がん）、胸部のがん（例えば、乳がん、肺がん（非小細胞肺がん、小細胞肺がん））、消化器のがん（例えば、食道がん、胃がん、十二指腸がん、大腸がん（結腸がん、直腸がん）、肝がん（肝細胞がん、胆管細胞がん）、胆嚢がん、胆管がん、膵がん、肛門がん）、泌尿器のがん（例えば、腎がん、尿管がん、膀胱がん、前立腺がん、陰茎がん、精巣（睾丸）がん）、生殖器のがん（例えば、子宮がん（子宮頸がん、子宮体がん）、卵巣がん、外陰がん、膣がん）、皮膚のがん（例えば、基底細胞がん、有棘細胞がん）を含むが、これらに限定されない。好ましくは、肺がん、膵がん、大腸がん、胃がん、食道がん、前立腺がん等が挙げられる。 The “cancer” to be subjected to the test method of the present invention is not particularly limited, and may be, for example, an epithelial cell-derived cancer, but may be non-epithelial sarcoma or hematologic cancer. More specifically, for example, cancer of the head and neck (eg, maxillary cancer, pharyngeal cancer, laryngeal cancer, tongue cancer, thyroid cancer), breast cancer (eg, breast cancer, lung cancer (non-small) Cell lung cancer, small cell lung cancer), digestive tract cancer (eg esophagus cancer, stomach cancer, duodenal cancer, colon cancer (colon cancer, rectum cancer), liver cancer (hepatocellular carcinoma, bile duct) Cell cancer), gallbladder cancer, bile duct cancer, pancreatic cancer, anal cancer), urinary cancer (eg, renal cancer, ureteral cancer, bladder cancer, prostate cancer, penile cancer, Testicular (testicular cancer), cancer of the genital organs (eg, uterine cancer (cervical cancer, uterine body cancer), ovarian cancer, vulvar cancer, vaginal cancer), skin cancer (eg, Include, but are not limited to basal cell carcinoma, squamous cell carcinoma). Preferably, lung cancer, pancreatic cancer, colon cancer, stomach cancer, esophagus cancer, prostate cancer and the like can be mentioned.
　本発明の検査方法の対象となる「線維症」は組織の線維化を伴う疾患であれば特に制限されず、例えば、肺線維症、肝線維症、肝硬変、非アルコール性脂肪肝炎（ＮＡＳＨ）、慢性Ｃ型肝炎、潰瘍性大腸炎、クローン病等が挙げられるが、これらに限定されない。また、本発明の検査方法は、前記の種々のがんにおける腫瘍間質の線維化の診断にも用いることができる。 The “fibrosis” to be subjected to the test method of the present invention is not particularly limited as long as it is a disease involving tissue fibrosis, and for example, pulmonary fibrosis, hepatic fibrosis, cirrhosis, nonalcoholic steatohepatitis (NASH), Chronic hepatitis C, ulcerative colitis, Crohn's disease and the like can be mentioned, but it is not limited thereto. In addition, the test method of the present invention can also be used for diagnosis of tumor stromal fibrosis in the above-mentioned various cancers.
　本明細書において「がん患者」、「線維症患者」とは、現在臨床的に用いられている確定診断法により、前記いずれかのがん、もしくは線維症に罹患していることが確認された者を意味し、がんもしくは線維症の「疑いのある者」とは、確定診断はなされていないが、何らかの検査、診察所見などにより、前記いずれかのがんもしくは線維症に罹患しているか、将来発症するおそれがあると予測される者を意味する。 In the present specification, “cancer patient” and “fibrosis patient” are confirmed to be suffering from any of the above-mentioned cancers or fibrosis by a definitive diagnostic method currently used clinically. The “suspected person” of cancer or fibrosis does not have a definitive diagnosis, but suffers from any of the above cancers or fibrosis by any test, medical examination, etc. It means a person who is predicted to have a risk of developing it in the future.
　本発明の検査方法に用いられる被検者由来の検体としては、本発明の抗体（１）及び（２）のいずれか一方又は両方を用いることで、がんもしくは線維症の診断、又はがんの悪性度もしくは線維化の進行度の診断が可能な検体であれば特に制限されず、例えば、生検組織、体液、エクソソーム等が挙げられる。体液としては、例えば、血液（例、全血、血清、血漿）、尿、腹水、膣洗浄液、唾液、脳脊髄液等が挙げられる。好ましくは血清、血漿等である。また、エクソソームとしては、前記の種々の体液中に含まれるエクソソームが挙げられる。好ましくは血清もしくは血漿から分離されたエクソソームである。エクソソームは、自体公知の超遠心沈降法、平衡密度勾配遠心法、免疫学的捕捉法、サイズ排除法、リン脂質アフィニティー、ポリマー沈降法などにより分離精製することができる。 As a sample derived from a subject used in the test method of the present invention, a diagnosis of cancer or fibrosis, or cancer by using either or both of the antibodies (1) and (2) of the present invention The sample is not particularly limited as long as it is a sample that can be diagnosed as having malignancy or progression of fibrosis, and examples thereof include biopsy tissue, body fluid, exosome and the like. Examples of the body fluid include blood (eg, whole blood, serum, plasma), urine, ascites fluid, vaginal lavage fluid, saliva, cerebrospinal fluid and the like. Preferably, it is serum, plasma or the like. Moreover, as exosome, the exosome contained in said various bodily fluid is mentioned. Preferably, they are exosomes separated from serum or plasma. The exosome can be separated and purified by ultracentrifugation, equilibrium density gradient centrifugation, immunological capture, size exclusion, phospholipid affinity, polymer precipitation, etc. which are known per se.
　上述のように、ＴＧＦ−β１はがん細胞の上皮間葉転換（ＥＭＴ）を促進することで、がん細胞に運動能を付与し、浸潤・転移に有利な特性を持たせる。また、ＴＧＦ−β１は、血管新生を促進することで、がん細胞の生存に寄与するとともに、転移ルートを提供する。さらに、腫瘍間質において、間葉系前駆細胞から線維芽細胞、筋線維芽細胞への分化を誘導し、がんの増殖・転移を促進する。また、がん細胞由来エクソソーム表面のＴＧＦ−β１がＣＴＬの活性化、ヘルパーＴ細胞やＮＫ細胞、マクロファージの機能を抑制し、がん防御免疫を無効化すると考えられている。
　上記の種々のＴＧＦ−β１の機能は、活性型ＴＧＦ−β１がＴＧＦ−β受容体を刺激することによって引き起こされるので、潜在型ＴＧＦ−β１ではこれらの機能を発現しないと考えられる。そのため、がん組織やその周辺組織における活性型及び潜在型ＴＧＦ−β１の各発現レベルを正確に測定することができれば、がんの悪性度（例、易転移性、易浸潤性）や線維化の進行度を鑑別することが可能となる。ところが、従来公知の抗ＴＧＦ−β１抗体は、後述の実施例において示されるとおり、検体（例えば、組織の種類）によって検出感度が非常に悪く、本来陽性であるべき検体でＴＧＦ−β１を検出できなかったり、あるいは、活性型ＴＧＦ−β１と潜在型ＴＧＦ−β１とを区別して検出することができないか、オフターゲットタンパク質と交差反応してしまう等、特異性にも問題があり、がんや線維症の診断にたえ得る抗体は存在しなかった。
　これに対し、本発明の抗体は、成熟ＴＧＦ−β１及びＬＡＰのそれぞれについて、進化的に保存されたユニークな部分アミノ酸配列を免疫原として作製されたものであるため、活性化型ＴＧＦ−β１又は潜在型ＴＧＦ−β１／ＬＡＰのいずれか一方のみを特異的に認識することができ、従って、検体中の活性化型ＴＧＦ−β１又は潜在型ＴＧＦ−β１／ＬＡＰの各発現レベルを正確に測定することが可能であり、また、オフターゲットタンパク質とも交差反応しないため、偽陽性のリスクが顕著に改善されている。しかも、従来公知の抗ＴＧＦ−β１抗体のほとんどでみられるような、検体による検出感度の低下（例、染色強度の低下）を認めず、活性化型ＴＧＦ−β１又は潜在型ＴＧＦ−β１／ＬＡＰを発現している任意の検体で、正しくいずれかのＴＧＦ−β１の発現を検出することができる。 As described above, TGF-β1 promotes epithelial-mesenchymal transition (EMT) of cancer cells, thereby imparting the cancer cells with motility and imparting advantageous properties to invasion and metastasis. In addition, TGF-β1 promotes angiogenesis to contribute to cancer cell survival and to provide a metastatic route. Furthermore, it induces differentiation of mesenchymal precursor cells into fibroblasts and myofibroblasts in tumor stroma, and promotes proliferation and metastasis of cancer. In addition, it is believed that TGF-β1 on the surface of cancer cell-derived exosomes activates CTL, suppresses the functions of helper T cells, NK cells, and macrophages, and invalidate cancer protective immunity.
It is considered that latent TGF-β1 does not express these functions because the various TGF-β1 functions mentioned above are triggered by stimulating active TGF-β1 with a TGF-β receptor. Therefore, if each expression level of active and latent TGF-β1 can be accurately measured in the cancer tissue and its surrounding tissue, the malignancy (eg, easy metastatic, easily invasive) or fibrosis of the cancer It is possible to distinguish the degree of progression of However, as shown in the following examples, conventionally known anti-TGF-β1 antibodies have very poor detection sensitivity depending on the sample (for example, the type of tissue) and can detect TGF-β1 in a sample that should be positive in nature Or there is a problem with specificity such as being unable to distinguish and detect active TGF-β1 and latent TGF-β1 or cross-reacting with off-target proteins, etc. There was no antibody that could be used to diagnose the disease.
On the other hand, since the antibody of the present invention is produced using the evolutionarily conserved unique partial amino acid sequence as an immunogen for each of mature TGF-β1 and LAP, activated TGF-β1 or Only one of latent TGF-β1 / LAP can be specifically recognized, and therefore, the expression level of each of activated TGF-β1 or latent TGF-β1 / LAP in a sample is accurately measured. The risk of false positives is significantly improved, as it is possible and does not cross react with off-target proteins. Furthermore, there is no reduction in the detection sensitivity (eg, reduction in staining intensity) by the sample as seen with most of the conventionally known anti-TGF-β1 antibodies, and activated TGF-β1 or latent TGF-β1 / LAP. The expression of any TGF-β1 can be detected correctly in any sample that is expressing
　本発明の抗体（１）及び／又は（２）を用いた、検体中の活性型ＴＧＦ−β１及び／又は潜在型ＴＧＦ−β１もしくはＬＡＰの検出は、その検体の種類に応じて、好適な抗原抗体反応アッセイを適宜選択することができる。例えば、検体が被検者から採取した生検組織の場合には、周知慣用の免疫組織染色を用いることができる。 Detection of active TGF-β1 and / or latent TGF-β1 or LAP in a sample using the antibody (1) and / or (2) of the present invention is a suitable antigen depending on the type of sample. An antibody response assay can be selected as appropriate. For example, in the case where the sample is a biopsy tissue collected from a subject, well-known conventional immunohistological staining can be used.
　標識物質を用いる測定法に用いられる標識剤としては、例えば、放射性同位元素、酵素、蛍光物質、発光物質などが用いられる。放射性同位元素としては、例えば、［１２５Ｉ］、［１３１Ｉ］、［３Ｈ］、［１４Ｃ］などが用いられる。上記酵素としては、安定で比活性の大きなものが好ましく、例えば、β−ガラクトシダーゼ、β−グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素などが用いられる。蛍光物質としては、例えば、フルオレスカミン、フルオレッセンイソチオシアネート（ＦＩＴＣ）、フィコエリスリン（ＰＥ）などが用いられる。発光物質としては、例えば、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなどが用いられる。 As a labeling agent used for a measurement method using a labeling substance, for example, radioactive isotopes, enzymes, fluorescent substances, luminescent substances and the like are used. As a radioactive isotope, for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used. As the above-mentioned enzyme, one which is stable and has a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate (FITC), phycoerythrin (PE) or the like is used. As the light-emitting substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
　本発明の抗体を直接標識物質で標識してもよいし、間接的に標識してもよい。好ましい態様においては、本発明の抗体は非標識抗体（一次抗体）とし、本発明の抗体を作製した動物に対する抗血清や抗Ｉｇ抗体等の標識された二次抗体により、活性型又は潜在型ＴＧＦ−β１を検出することができる。あるいは、ビオチン化した二次抗体を用いて、ＴＧＦ−β１−本発明の抗体−二次抗体の複合体を形成させ、これを標識したストレプトアビジンを用いて可視化することもできる。 The antibody of the present invention may be directly labeled with a labeling substance, or may be labeled indirectly. In a preferred embodiment, the antibody of the present invention is a non-labeled antibody (primary antibody), and a labeled secondary antibody such as an antiserum or an anti-Ig antibody to an animal in which the antibody of the present invention is produced -Β1 can be detected. Alternatively, a biotinylated secondary antibody can be used to form a complex of TGF-.beta.1-antibody-secondary antibody of the present invention, which can be visualized using labeled streptavidin.
　例えば、生検組織サンプルをグルタルアルデヒド、パラホルムアルデヒド等で固定・透過処理しＰＢＳ等の緩衝液で洗浄、ＢＳＡ等でブロッキングした後、本発明の抗体とインキュベートする。ＰＢＳ等の緩衝液で洗浄して未反応の抗体を除去した後、本発明のと反応した組織を標識した二次抗体で可視化し、顕微鏡下で目視観察したり、共焦点レーザー走査型顕微鏡や、ＩＮ　Ｃｅｌｌ　Ａｎａｌｙｚｅｒ（Ａｍａｒｓｈａｍ／ＧＥ）等の自動化された生細胞画像解析装置等を用いて定量解析することができる。 For example, a biopsy tissue sample is fixed / permeabilized with glutaraldehyde, paraformaldehyde or the like, washed with a buffer such as PBS, blocked with BSA or the like, and then incubated with the antibody of the present invention. After washing with a buffer solution such as PBS to remove unreacted antibody, the tissue reacted with the present invention is visualized with a labeled secondary antibody, and visually observed under a microscope, or a confocal laser scanning microscope or Quantitative analysis using an automated live cell image analyzer such as IN Cell Analyzer (Amarsham / GE).
　例えば、本発明の抗体を用いてがん組織とその周辺の正常組織の標本サンプルを免疫染色した場合、本発明の抗体（１）及び（２）のいずれによっても、がん組織、良性組織（例、腺組織、筋組織、線維芽細胞等）のどちらも染色されるが、典型的には、本発明の抗体（１）では、がん組織や、良性組織であっても炎症の強い部位や線維化が著明な部位では、強く染色され、良性組織では染色性が比較的弱いのに対し、本発明の抗体（２）では、がん組織の染色性は本発明の抗体（１）に比べて弱く、良性組織においては、本発明の抗体（１）と同等もしくはそれ以上の染色性を示す。即ち、がん組織では活性型ＴＧＦ−β１が高発現している一方で、良性組織では潜在型ＴＧＦ−β１の発現が比較的高いと考えられる。がん組織やその周辺組織（特に、炎症の強い部位や線維化の目立つ部位）で活性型ＴＧＦ−β１の発現が潜在型ＴＧＦ−β１の発現と比較してより顕著である場合には、被検者のがんは悪性度が高い（例えば、遠隔転移しやすい、周辺組織に浸潤しやすい）と予測することができる。あるいはまた、腫瘍間質において線維芽細胞や筋線維芽細胞への分化が促進され、当該部位において潜在型及び／又は活性型ＴＧＦ−β１の高発現が認められる場合には、被検者は腫瘍随伴線維症を発症していると予測することができる。被検者が線維症患者又はその疑いのある者の場合でも、同様に、検体組織において潜在型及び／又は活性型ＴＧＦ−β１の高発現が認められる場合には、該被検者は線維症を発症しているか将来発症するリスクが高い、あるいは線維化が進行している等と予測することができる。 For example, when a sample of a cancer tissue and a normal tissue in the vicinity thereof is immunostained using the antibody of the present invention, cancer tissues and benign tissues (both by the antibodies (1) and (2) of the present invention) For example, glandular tissue, muscle tissue, fibroblast etc.) are both stained, but typically, with the antibody (1) of the present invention, a site of strong inflammation even in cancer tissue or benign tissue And in areas where fibrosis is prominent, staining is strong in benign tissues and is relatively weak in benign tissues, whereas in the case of the antibody (2) of the present invention, the staining property of cancerous tissue is the antibody (1) of the present invention It is weaker than the above, and in benign tissues, it shows the staining property equal to or more than that of the antibody (1) of the present invention. That is, in cancer tissues, active TGF-β1 is highly expressed, while in benign tissues, expression of latent TGF-β1 is considered to be relatively high. If the expression of active TGF-β1 is more pronounced in cancer tissues and their surrounding tissues (in particular, sites of strong inflammation and sites of marked fibrosis) compared to the expression of latent TGF-β1, The examiner's cancer can be predicted to be of high grade (eg, prone to distant metastasis, prone to invade surrounding tissues). Alternatively, if differentiation into fibroblasts or myofibroblasts is promoted in the tumor stroma and high expression of latent and / or active TGF-β1 is observed at the relevant site, the subject is a tumor It can be predicted that associated fibrosis is developing. Even if the subject is a fibrotic patient or a person suspected of having the same, the subject is also fibrotic if high expression of latent and / or active TGF-β1 is observed in the sample tissue. It can be predicted that there is a high risk of developing or a future onset of fibrosis, or that fibrosis is progressing.
　染色性の比較は、検体組織の病変部と非病変部との間でなされてもよいし、同一被検者から時系列的に採取された検体間でなされてもよい。あるいは、被検者がんもしくは線維症の疑いのある者の場合には、健常者由来の対応する検体との比較により、発症の有無又は発症リスクの判定を行うこともできる。 The comparison of staining property may be made between the lesioned part and non-lesioned part of the sample tissue, or may be made between samples collected in time series from the same subject. Alternatively, in the case of a subject who is suspected of having cancer or fibrosis, the presence or absence of onset or the onset risk can be determined by comparison with a corresponding sample derived from a healthy subject.
　また、悪性度が高い転移性のがんにあっては、がん細胞から分泌されたＴＧＦ−β１が血液をはじめとする体液中に漏出し、また、がん細胞由来のエクソソームの膜表面に包埋されて体液中に輸送されるが、血中やエクソソーム中に活性型ＴＧＦ−β１の発現レベルががんの悪性度と相関すると考えられるので、これらの検体中の活性型ＴＧＦ−β１の発現レベルを、本発明の抗体（１）を用いて測定し、コントロール（例、非転移性がん患者由来の検体）と比較するか、あるいは同一検体における潜在型ＴＧＦ−β１の発現レベルを本発明の抗体（２）を用いて測定し、活性型ＴＧＦ−β１と潜在型ＴＧＦ−β１の存在比を比較することにより、被検者におけるがんの悪性度を予測することができる。同様に、線維症の進行度を予測することもできる。
　血中ＴＧＦ−β１の測定には、自体公知のＥＬＩＳＡや蛍光免疫アッセイ等の各種イムノアッセイを用いることができる。あるいは、血清もしくは血漿から常法によりタンパク質を抽出し、ウェスタンブロット解析によりＴＧＦ−β１レベルを測定することもできる。 In addition, in high-grade metastatic cancer, TGF-β1 secreted from cancer cells leaks into blood and other body fluids, and on the membrane surface of cancer cell-derived exosomes. Although it is embedded and transported into body fluid, the expression level of active TGF-β1 in blood and exosomes is considered to be correlated with the malignancy of cancer, so that active TGF-β1 in these samples is The expression level is measured using the antibody (1) of the present invention, and compared with a control (eg, a sample derived from a non-metastatic cancer patient), or the expression level of latent TGF-β1 in the same sample is The malignancy of a cancer in a subject can be predicted by measuring using the antibody (2) of the invention and comparing the abundance ratio of active TGF-β1 and latent TGF-β1. Similarly, the degree of progression of fibrosis can also be predicted.
Various immunoassays such as ELISA and fluorescence immunoassay known per se can be used to measure blood TGF-β1. Alternatively, proteins can be extracted from serum or plasma by a conventional method, and TGF-β1 levels can be measured by Western blot analysis.
　一方、エクソソーム表面に存在するＴＧＦ−β１の測定は、例えば、本発明の抗体を磁性ビーズ上に固定化し、磁場を与えることでＴＧＦ−β１を表面に提示するエクソソームがサンプルから分離（即ち、磁気活性化細胞分離（ＭＡＣＳ））するようにすることができる。別の好ましい態様においては、本発明の抗体を、上述のような任意の適切な蛍光分子で直接もしくは間接的に標識し、蛍光活性化セルソーター（ＦＡＣＳ）を用いてＴＧＦ−β１を表面に提示するエクソソームを単離することができ、該標識の強度を指標として、ＴＧＦ−β１レベルを測定することができる。 On the other hand, for measurement of TGF-β1 present on the exosome surface, for example, an antibody of the present invention is immobilized on magnetic beads, and an exosome presenting TGF-β1 on the surface is separated from the sample by applying a magnetic field (ie, magnetic It can be made to activate cell separation (MACS). In another preferred embodiment, the antibody of the present invention is directly or indirectly labeled with any suitable fluorescent molecule as described above, and a TGF-activated cell sorter (FACS) is used to display TGF-β1 on the surface Exosomes can be isolated, and TGF-β1 levels can be measured using the intensity of the label as an index.
　あるいは、上記の血中もしくはエクソソーム表面のＴＧＦ−β１測定において、本発明の抗体（２）を用いて潜在型ＴＧＦ−β１のレベルを調べるか、本発明の抗体（１）と組み合わせて活性型ＴＧＦ−β１のレベルと比較することにより、がんのより早期の病態を検出することも可能となる。同様に、線維症の早期診断にも適用可能である。 Alternatively, in the above-mentioned TGF-β1 measurement on blood or exosome surface, the level of latent TGF-β1 is examined using the antibody (2) of the present invention, or activated TGF in combination with the antibody (1) of the present invention By comparing with the level of β1, it is also possible to detect an earlier disease state of cancer. Likewise, it is applicable to early diagnosis of fibrosis.
　また、がん又は線維症患者の治療前後で検体を採取し、同様の検査を実施し、その結果を比較することにより、当該治療の奏効性を評価することもできる。 In addition, it is also possible to evaluate the efficacy of the treatment by collecting a sample before and after treatment of a cancer or fibrotic patient, performing the same test, and comparing the results.
［ＩＩＩ］本発明のキット
　本発明はまた、本発明の抗体（１）及び本発明の抗体（２）を含んでなる、がんもしくは線維症の診断、又はがんの悪性度もしくは線維化の進行度の診断用キットを提供する。当該キットは、上記した本発明の検査方法の実施に適した、他の自体公知の構成成分をさらに含んでもよい。 [III] Kit of the Present Invention The present invention also provides a diagnosis of cancer or fibrosis, or cancer malignancy or fibrosis comprising the antibody (1) of the present invention and the antibody (2) of the present invention Provide a diagnostic kit for progression. The kit may further contain other known components suitable for carrying out the above-described test method of the present invention.
　以下、実施例により本発明をさらに詳しく説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.
実施例１　本発明の抗体の作製
（１）免疫原の調製
　潜在型ＴＧＦ−β１ポリペプチドのＬＡＰ及び成熟ＴＧＦ−β１領域のそれぞれに位置する進化的に保存されたユニークな配列（１０アミノ酸）を、商業的に利用可能なエピトープ予測ソフトウェア「Ｅｐｉｔｏｐｅ　Ｈｕｎｔｅｒ」を用いて抽出した。抽出された配列からなるペプチドをペプチド自動合成機を用いて合成し、それらを担体に連結して免疫原とした。 Example 1 Preparation of Antibody of the Present Invention (1) Preparation of Immunogen An evolutionarily conserved unique sequence (10 amino acids) located in each of LAP and mature TGF-β1 region of latent TGF-β1 polypeptide It was extracted using commercially available epitope prediction software "Epitope Hunter". Peptides consisting of the extracted sequences were synthesized using an automatic peptide synthesizer, and they were linked to a carrier to make an immunogen.
（２）免疫感作
　上記（１）で得られた免疫原を等量の完全フロイントアジュバント又は不完全フロイントアジュバントとともに乳化し、マウスに腹腔内注射した。その後、追加免疫を行い、マウスを安楽死させた。 (2) Immunization The immunogen obtained in the above (1) was emulsified with an equal amount of complete Freund's adjuvant or incomplete Freund's adjuvant and injected intraperitoneally into mice. Thereafter, a boost was given to euthanize the mice.
（３）細胞融合、クローニング及びアッセイ
　免疫したマウスのうち、血清の間接ＥＬＩＳＡで高力価を示したものの脾臓からリンパ球を単離し、ポリエチレングリコールを用いてマウスミエローマ細胞と融合した。融合細胞を９６ウェル組織培養プレートに播種し、ハイブリドーマ用培地を添加してハイブリドーマ（６４クローン）を選択した。選択はＥＬＩＳＡ試験により実施した。各ハイブリドーマの培養上清を、抗原ペプチド又はＴＧＦ−β１でコーティングしたスクリーニングプレート（ＢＳＡ含有ブロッキング液で前処理済）に添加し、室温にてインキュベートした。ＰＢＳでプレートを洗浄後、１；１５００希釈した西洋ワサビペルオキシダーゼ（ＨＲＰ）標識したヤギ抗マウスＩｇＧを各ウェルに添加してインキュベートした。プレートを洗浄後、発色基質（ＯＰＤ　ｓｙｓｔｅｍ）を加えて５分間発色させた後、４５０ｎｍにおける吸光度を測定した。６４クローンすべてが抗原ペプチドをコーティングした間接ＥＬＩＳＡで陽性であった。それらのうち１１クローンはＴＧＦ−β１をコーティングした間接ＥＬＩＳＡでも陽性であった。
　次に、この１１クローンをウェスタンブロット解析に供した。ＴＧＦ−β１を過剰発現する細胞又は組織ライセートをＳＤＳ−ポリアクリルアミド電気泳動にかけ、５倍希釈したハイブリドーマ培養上清と反応させ、免疫反応性をＨＲＰ標識ヤギ抗マウスＩｇＧを用いて可視化した。１１クローン中１０クローンが、細胞及び組織ライセートの両方に対し陽性であった。
　さらに、ＨｅｐＧ２細胞に対する蛍光イムノアッセイを行った。１０クローン中６クローンが強い陽性シグナルを示した。これらのうち２クローン（２Ｈ４及び４Ｄ１０）を用いて、さらに詳細な解析を行った。
　同様にして、ＬＡＰの部分ペプチドで免疫したマウスから、抗潜在型ＴＧＦ−β１／ＬＡＰモノクローナル抗体クローン（２Ｆ１０）を得た。 (3) Cell fusion, cloning and assay Among the immunized mice, those showing high titer by indirect ELISA of serum were isolated from the spleen of the lymphocytes and fused with the mouse myeloma cells using polyethylene glycol. The fused cells were seeded in 96 well tissue culture plates and hybridoma culture medium was added to select hybridomas (64 clones). Selection was performed by ELISA test. The culture supernatant of each hybridoma was added to a screening plate (pretreated with a BSA-containing blocking solution) coated with an antigenic peptide or TGF-β1, and incubated at room temperature. After washing the plate with PBS, 1; 1500 diluted horseradish peroxidase (HRP) labeled goat anti-mouse IgG was added to each well and incubated. After washing the plate, a chromogenic substrate (OPD system) was added to develop color for 5 minutes, and then the absorbance at 450 nm was measured. All 64 clones were positive in the indirect ELISA coated with antigenic peptide. Among them, 11 clones were also positive in the indirect ELISA coated with TGF-β1.
Next, the 11 clones were subjected to Western blot analysis. Cells or tissue lysates overexpressing TGF-.beta.1 were subjected to SDS-polyacrylamide electrophoresis, reacted with 5-fold diluted hybridoma culture supernatant, and immunoreactivity was visualized using HRP-labeled goat anti-mouse IgG. Ten out of eleven clones were positive for both cell and tissue lysates.
In addition, fluorescent immunoassay was performed on HepG2 cells. Six out of ten clones showed a strong positive signal. Further detailed analysis was performed using 2 clones (2H4 and 4D10) of these.
Similarly, anti-latent TGF-β1 / LAP monoclonal antibody clone (2F10) was obtained from mice immunized with a partial peptide of LAP.
実施例２　抗活性型ＴＧＦ−β１抗体及び抗潜在型ＴＧＦ−β１／ＬＡＰ抗体を用いた大腸がん組織標本の免疫染色
（１）使用した試薬、キット
・ＶＥＣＴＡＳＴＡＩＮ　ＡＢＣ　ＫＩＴ　Ｍｏｕｓｅ　ＩｇＧ（ＰＫ−４００２）
・ＩｍｍＰＡＣＴ　ＤＡＢ　Ｐｅｒｏｘｉｄａｓｅ　Ｓｕｂｓｔｒａｔｅ　Ｋｉｔ（ＳＫ−４１０５）
・各種一次抗体（実施例１で作製した抗体、並びにＳａｎｔａ　Ｃｒｕｚ社、Ｎｏｕｓ　Ｂｉｏ社、ＬＳ　Ｂｉｏ社、ＰｅｐｒｏＴｅｃｈ社、Ｓｉｇｎａｌ社及びＲ＆Ｄ社製の市販抗体）
・過酸化水素水、メタノール
・ＰＢＳ
・クエン酸ナトリウム、クエン酸三ナトリウム二水和物
・Ｘｙｌｏｌ、Ｅｔｈａｎｏｌ
・Ｍａｙｅｒ’ｓ　Ｈｅｍａｔｏｘｙｌｉｎ Example 2 Immunostaining of colorectal cancer tissue specimens using anti-active TGF-β1 antibody and anti-latent TGF-β1 / LAP antibody (1) Reagents used, kit VECTASTAIN ABC KIT Mouse IgG (PK-4002)
・ ImmPACT DAB Peroxidase Substrate Kit (SK-4105)
・ Various primary antibodies (antibodies prepared in Example 1 and commercially available antibodies manufactured by Santa Cruz, Nous Bio, LS Bio, PeproTech, Signal and R & D)
· Hydrogen peroxide water, methanol · PBS
Sodium citrate, trisodium citrate dihydrate Xylol, Ethanol
Mayer's Hematoxylin
Figure JPOXMLDOC01-appb-I000001
Figure JPOXMLDOC01-appb-I000001
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000003
Figure JPOXMLDOC01-appb-I000003
（４）本発明の抗潜在型ＴＧＦ−β１／ＬＡＰ抗体と市販の各種抗ＴＧＦ−β１抗体との比較
　本発明の抗潜在型ＴＧＦ−β１／ＬＡＰ抗体（２Ｆ１０）、又は市販の各種抗ＴＧＦ−β１抗体を一次抗体として、大腸がんの組織切片の免疫染色を行った。結果を図１及び２、表１に示す。 (4) Comparison of the anti-latent TGF-β1 / LAP antibody of the present invention with various commercially available anti-TGF-β1 antibodies The anti-latent TGF-β1 / LAP antibody (2F10) of the present invention, or various commercially available anti-TGF- Immunostaining of colorectal cancer tissue sections was performed using the β1 antibody as a primary antibody. The results are shown in FIGS.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　比較例として用いた６種の市販抗体は、いずれもＴＧＦ−β１を染色可能であるとしているが、ＰｅｐｒｏＴｅｃｈ社製の抗体以外の抗体を用いた場合、がん組織でＴＧＦ−β１を検出できなかった。一方、実施例１で得られた本発明の抗潜在型ＴＧＦ−β１／ＬＡＰ抗体を一次抗体として用いた場合は、がん組織でもＴＧＦ−β１を検出することができた。ＴＧＦ−β１はがん以外に線維化にも関与しており、線維化と深く関連する線維芽細胞、筋線維芽細胞でもＴＧＦ−β１が検出された。 All six commercially available antibodies used as comparative examples are supposed to be capable of staining TGF-β1, but TGF-β1 can not be detected in cancer tissues when using an antibody other than PeproTech's antibody. The On the other hand, when the anti-latent TGF-β1 / LAP antibody of the present invention obtained in Example 1 was used as a primary antibody, TGF-β1 could be detected even in cancer tissues. TGF-.beta.1 is also involved in fibrosis other than cancer, and fibroblasts and myofibroblasts, which are closely related to fibrosis, were also detected in TGF-.beta.1.
（５）本発明の抗潜在型ＴＧＦ−β１／ＬＡＰ抗体と抗活性型ＴＧＦ−β１抗体との比較
　上記（４）と同様にして、実施例１で得られた本発明の抗潜在型ＴＧＦ−β１／ＬＡＰ抗体（２Ｆ１０）又は抗活性型ＴＧＦ−β１抗体（２Ｈ４）を一次抗体として用い、大腸がん組織切片の免疫染色を行った。結果を図３に示す。がん組織では、活性型ＴＧＦ−β１が強く発現していた。一方、良性組織でも炎症の強い部位や線維化の目立つ部位では、炎症細胞や線維芽細胞で活性型及び潜在型ＴＧＦ−β１とも比較的強く発現していた。抗活性型ＴＧＦ−β１抗体として４Ｄ１０を用いた場合でも、同様に、がん組織における活性型ＴＧＦ−β１の強発現が観察された（図４）。 (5) Comparison of the anti-latent TGF-β1 / LAP antibody of the present invention with the anti-active TGF-β1 antibody In the same manner as in (4) above, the anti-latent TGF- of the present invention obtained in Example 1 Immunostaining of colon cancer tissue sections was performed using β1 / LAP antibody (2F10) or anti-active TGF-β1 antibody (2H4) as a primary antibody. The results are shown in FIG. In cancer tissues, active TGF-β1 was strongly expressed. On the other hand, even in benign tissues, in areas with strong inflammation and areas with marked fibrosis, both active and latent TGF-β1 were relatively strongly expressed in inflammatory cells and fibroblasts. Even when 4D10 was used as the anti-active TGF-β1 antibody, strong expression of active TGF-β1 in cancer tissues was similarly observed (FIG. 4).
　本発明を好ましい態様を強調して説明してきたが、好ましい態様が変更され得ることは当業者にとって自明であろう。本発明は、本発明が本明細書に詳細に記載された以外の方法で実施され得ることを意図する。したがって、本発明は添付の「請求の範囲」の精神及び範囲に包含されるすべての変更を含むものである。
　ここで述べられた特許及び特許出願明細書を含む全ての刊行物に記載された内容は、ここに引用されたことによって、その全てが明示されたと同程度に本明細書に組み込まれるものである。 Although the invention has been described with emphasis on preferred embodiments, it will be obvious to those skilled in the art that the preferred embodiments can be varied. The present invention contemplates that the present invention may be practiced in ways other than as specifically described herein. Accordingly, the present invention is intended to cover all modifications that fall within the spirit and scope of the appended claims.
The contents described in all the publications, including the patents and patent applications mentioned here, are hereby incorporated by reference in their entirety to the same extent as if expressly set forth. .
　本出願は、２０１７年８月２９日付で日本国に出願された特願２０１７−１６４８８９を基礎としており、ここで言及することによりその内容は全て本明細書に包含される。 This application is based on patent application No. 2017-164889 filed in Japan on August 29, 2017, the entire contents of which are incorporated herein by reference.
　本発明によれば、腫瘍の悪性度（遠隔転移しやすいか、周辺組織に浸潤しやすいか）の判断、治療薬の薬効評価が可能になる。また、血中やエクソソーム表面のＴＧＦ−β１レベルを測定し、活性型と潜在型の発現を比較することによってもがんの悪性度の判断、早期がん診断、予後予測などが可能となる。さらに、腫瘍に伴う線維症や全身の線維症の診断も可能となる。よって、本発明の検査方法及び検査用キットは、臨床上きわめて有用である。
According to the present invention, it is possible to determine the grade of a tumor (whether it is likely to be distant metastasis or to invade surrounding tissues) and to evaluate the efficacy of a therapeutic agent. Also, by measuring TGF-β1 levels in the blood or exosome surface and comparing the expression of active and latent forms, judgment of malignancy of cancer, early cancer diagnosis, prognosis prediction, etc. become possible. In addition, diagnosis of fibrosis associated with tumor and systemic fibrosis is also possible. Therefore, the test method and test kit of the present invention are extremely useful clinically.

Claims (8)

  1. 　がんもしくは線維症患者、又はその疑いのある者由来の検体と、
    　以下の（ａ１）~（ｆ１）の相補性決定領域（ＣＤＲ）：
    （ａ１）配列番号１で表されるアミノ酸配列を含むＣＤＲ、
    （ｂ１）配列番号２で表されるアミノ酸配列を含むＣＤＲ、
    （ｃ１）配列番号３で表されるアミノ酸配列を含むＣＤＲ、
    （ｄ１）配列番号４で表されるアミノ酸配列を含むＣＤＲ、
    （ｅ１）配列番号５で表されるアミノ酸配列を含むＣＤＲ、及び
    （ｆ１）配列番号６で表されるアミノ酸配列を含むＣＤＲ
    を含み、活性型ＴＧＦ−β１に結合するが、潜在型ＴＧＦ−β１には結合しない第１のモノクローナル抗体、及び／又は
    　以下の（ａ２）~（ｆ２）のＣＤＲ：
    （ａ２）配列番号７で表されるアミノ酸配列を含むＣＤＲ、
    （ｂ２）配列番号８で表されるアミノ酸配列を含むＣＤＲ、
    （ｃ２）配列番号９で表されるアミノ酸配列を含むＣＤＲ、
    （ｄ２）配列番号１０で表されるアミノ酸配列を含むＣＤＲ、
    （ｅ２）配列番号１１で表されるアミノ酸配列を含むＣＤＲ、及び
    （ｆ２）配列番号１２で表されるアミノ酸配列を含むＣＤＲ
    を含み、潜在型ＴＧＦ−β１及びＬＡＰに結合するが、活性型ＴＧＦ−β１には結合しない第２のモノクローナル抗体
    とを接触させ、該検体中の活性型ＴＧＦ−β１及び／又は潜在型ＴＧＦ−β１もしくはＬＡＰを特異的に検出することを特徴とする、がんもしくは線維症の診断、又はがんの悪性度もしくは線維化の進行度の診断のための検査方法。     A sample from a patient with cancer or fibrosis or who is suspected of
    The following (a1) to (f1) complementarity determining regions (CDRs):
    (A1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 1,
    (B1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 2,
    (C1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 3,
    (D1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 4,
    (E1) CDR comprising the amino acid sequence represented by SEQ ID NO: 5, and (f1) CDR comprising the amino acid sequence represented by SEQ ID NO: 6
    A first monoclonal antibody that binds to active TGF-β1 but not to latent TGF-β1, and / or CDRs of (a2) to (f2) below:
    (A2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 7,
    (B2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 8,
    (C2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 9,
    (D2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 10,
    (E2) CDR comprising the amino acid sequence represented by SEQ ID NO: 11 and (f2) CDR comprising the amino acid sequence represented by SEQ ID NO: 12
    Contact with a second monoclonal antibody that contains latent TGF-.beta.1 and LAP but does not bind to active TGF-.beta.1, active TGF-.beta.1 in the sample and / or latent TGF-.beta. A test method for diagnosing cancer or fibrosis, or diagnosing cancer malignancy or progression of fibrosis, which is characterized by specifically detecting β1 or LAP.
  2. 　第１のモノクローナル抗体の重鎖可変領域に含まれるＣＤＲが上記（ａ１）、（ｂ１）及び（ｃ１）であり、軽鎖可変領域に含まれるＣＤＲが上記（ｄ１）、（ｅ１）及び（ｆ１）であり、
    　第２のモノクローナル抗体の重鎖可変領域に含まれるＣＤＲが上記（ａ２）、（ｂ２）及び（ｃ２）であり、軽鎖可変領域に含まれるＣＤＲが上記（ｄ２）、（ｅ２）及び（ｆ２）である、
    請求項１に記載の方法。     The CDRs contained in the heavy chain variable region of the first monoclonal antibody are the above (a1), (b1) and (c1), and the CDRs contained in the light chain variable region are the above (d1), (e1) and (f1) ) And
    The CDRs contained in the heavy chain variable region of the second monoclonal antibody are the above (a2), (b2) and (c2), and the CDRs contained in the light chain variable region are the above (d2), (e2) and (f2) ),
    The method of claim 1.
  3. 　第１のモノクローナル抗体の重鎖可変領域に含まれるＣＤＲ１、ＣＤＲ２及びＣＤＲ３がそれぞれ上記（ａ１）、（ｂ１）及び（ｃ１）であり、軽鎖可変領域に含まれるＣＤＲ１、ＣＤＲ２及びＣＤＲ３がそれぞれ上記（ｄ１）、（ｅ１）及び（ｆ１）であり、
    　第２のモノクローナル抗体の重鎖可変領域に含まれるＣＤＲ１、ＣＤＲ２及びＣＤＲ３がそれぞれ上記（ａ２）、（ｂ２）及び（ｃ２）であり、軽鎖可変領域に含まれるＣＤＲ１、ＣＤＲ２及びＣＤＲ３がそれぞれ上記（ｄ２）、（ｅ２）及び（ｆ２）である、
    請求項１に記載の方法。     The CDR1, CDR2 and CDR3 contained in the heavy chain variable region of the first monoclonal antibody are the above (a1), (b1) and (c1) respectively, and the CDR1, CDR2 and CDR3 contained in the light chain variable region are each mentioned above (D1), (e1) and (f1),
    The CDR1, CDR2 and CDR3 contained in the heavy chain variable region of the second monoclonal antibody are the above (a2), (b2) and (c2) respectively, and the CDR1, CDR2 and CDR3 contained in the light chain variable region are each mentioned above (D2), (e2) and (f2),
    The method of claim 1.
  4. 　第１のモノクローナル抗体が、
    （Ｘ１）配列番号１３で表されるアミノ酸配列を含む重鎖可変領域、及び
    （Ｙ１）配列番号１４で表されるアミノ酸配列を含む軽鎖可変領域
    を含み、
    　第２のモノクローナル抗体が、
    （Ｘ２）配列番号１５で表されるアミノ酸配列を含む重鎖可変領域、及び
    （Ｙ２）配列番号１６で表されるアミノ酸配列を含む軽鎖可変領域
    を含む、請求項１に記載の方法。     The first monoclonal antibody is
    (X1) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13; and (Y1) a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14;
    The second monoclonal antibody is
    The method according to claim 1, comprising (X2) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15, and (Y2) a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16.
  5. 　第１のモノクローナル抗体が、請求項１~４のいずれか１項に記載の第１のモノクローナル抗体と競合的に活性型ＴＧＦ−β１に結合し、かつ潜在型ＴＧＦ−β１には結合しない抗体であり、
    　第２のモノクローナル抗体が、請求項１~４のいずれか１項に記載の第２のモノクローナル抗体と競合的に潜在型ＴＧＦ−β１もしくはＬＡＰに結合し、かつ活性型ＴＧＦ−β１には結合しない抗体である、請求項１~４のいずれか１項に記載の方法。     The first monoclonal antibody is an antibody that binds to active TGF-β1 competitively with the first monoclonal antibody according to any one of claims 1 to 4 and does not bind to latent TGF-β1. Yes,
    The second monoclonal antibody binds to latent TGF-β1 or LAP competitively with the second monoclonal antibody according to any one of claims 1 to 4 and does not bind to active TGF-β1 The method according to any one of claims 1 to 4, which is an antibody.
  6. 　検体が生検組織、体液又はエクソソームである、請求項１~５のいずれか１項に記載の方法。 6. The method according to any one of claims 1 to 5, wherein the sample is a biopsy tissue, a body fluid or an exosome.
  7. 　検出法が、免疫組織染色、ＥＬＩＳＡ、ウェスタンブロット、ＦＡＣＳ及びＭＡＣＳからなる群より選択される、請求項１~６のいずれか１項に記載の方法。 7. The method according to any one of claims 1 to 6, wherein the detection method is selected from the group consisting of immunohistological staining, ELISA, western blot, FACS and MACS.
  8. 　以下の（ａ１）~（ｆ１）の相補性決定領域（ＣＤＲ）：
    （ａ１）配列番号１で表されるアミノ酸配列を含むＣＤＲ、
    （ｂ１）配列番号２で表されるアミノ酸配列を含むＣＤＲ、
    （ｃ１）配列番号３で表されるアミノ酸配列を含むＣＤＲ、
    （ｄ１）配列番号４で表されるアミノ酸配列を含むＣＤＲ、
    （ｅ１）配列番号５で表されるアミノ酸配列を含むＣＤＲ、及び
    （ｆ１）配列番号６で表されるアミノ酸配列を含むＣＤＲ
    を含み、活性型ＴＧＦ−β１に結合するが、潜在型ＴＧＦ−β１には結合しない第１のモノクローナル抗体、及び
    　以下の（ａ２）~（ｆ２）のＣＤＲ：
    （ａ２）配列番号７で表されるアミノ酸配列を含むＣＤＲ、
    （ｂ２）配列番号８で表されるアミノ酸配列を含むＣＤＲ、
    （ｃ２）配列番号９で表されるアミノ酸配列を含むＣＤＲ、
    （ｄ２）配列番号１０で表されるアミノ酸配列を含むＣＤＲ、
    （ｅ２）配列番号１１で表されるアミノ酸配列を含むＣＤＲ、及び
    （ｆ２）配列番号１２で表されるアミノ酸配列を含むＣＤＲ
    を含み、潜在型ＴＧＦ−β１及びＬＡＰに結合するが、活性型ＴＧＦ−β１には結合しない第２のモノクローナル抗体を含んでなる、がんもしくは線維症の診断、又はがんの悪性度もしくは線維化の進行度の診断用キット。     The following (a1) to (f1) complementarity determining regions (CDRs):
    (A1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 1,
    (B1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 2,
    (C1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 3,
    (D1) a CDR comprising the amino acid sequence represented by SEQ ID NO: 4,
    (E1) CDR comprising the amino acid sequence represented by SEQ ID NO: 5, and (f1) CDR comprising the amino acid sequence represented by SEQ ID NO: 6
    And a first monoclonal antibody that binds to active TGF-β1 but not latent TGF-β1, and the CDRs of (a2) to (f2) below:
    (A2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 7,
    (B2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 8,
    (C2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 9,
    (D2) a CDR comprising the amino acid sequence represented by SEQ ID NO: 10,
    (E2) CDR comprising the amino acid sequence represented by SEQ ID NO: 11 and (f2) CDR comprising the amino acid sequence represented by SEQ ID NO: 12
    Diagnosis of cancer or fibrosis, or cancer grade or fiber comprising a second monoclonal antibody comprising: TGF-.beta.1 and LAP but binding to latent TGF-.beta.1 but not to active TGF-.beta.1 Diagnostic kit for
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