WO2019045086A1 - UTILISATION D'UN ANTICORPS SPÉCIFIQUE POUR TGF-β1 ACTIF OU LATENT - Google Patents

UTILISATION D'UN ANTICORPS SPÉCIFIQUE POUR TGF-β1 ACTIF OU LATENT 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

La présente invention concerne un procédé d'examen pour diagnostiquer un cancer ou une fibrose ou pour diagnostiquer une malignité cancéreuse ou une progression de fibrose, ledit procédé d'examen étant caractérisé en ce qu'un échantillon provenant d'une personne qui a ou peut avoir un cancer ou une fibrose est amené à entrer en contact avec un premier anticorps monoclonal, qui se lie au TGF-β1 actif mais ne se lie pas au TGF-β1 latent et comprend (a1) une région de détermination de complémentarité (CDR) contenant la séquence d'acides aminés représentée par le numéro de séquence 1, (b1) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 2, (c1) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 3, (d1) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 4, (e1) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 5, et (f1) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 6, et/ou un deuxième anticorps monoclonal, qui se lie au TGF-β1 latent et au LAP mais ne se lie pas au TGF-β1 actif et comprend (a2) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 7, (b2) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 8, (c2) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 9, (d2) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 10, (e2) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 11, et (f2) un CDR contenant la séquence d'acides aminés représentée par le numéro de séquence 12, et le TGF-β1 actif et/ou le TGF-β1 latent ou le LAP latent dans l'échantillon est détecté spécifiquement.
PCT/JP2018/032514 2017-08-29 2018-08-28 UTILISATION D'UN ANTICORPS SPÉCIFIQUE POUR TGF-β1 ACTIF OU LATENT WO2019045086A1 (fr)

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