WO2018235964A1 - Anticorps anti-tgf-bêeta1 - Google Patents

Anticorps anti-tgf-bêeta1 Download PDF

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Publication number
WO2018235964A1
WO2018235964A1 PCT/JP2018/023920 JP2018023920W WO2018235964A1 WO 2018235964 A1 WO2018235964 A1 WO 2018235964A1 JP 2018023920 W JP2018023920 W JP 2018023920W WO 2018235964 A1 WO2018235964 A1 WO 2018235964A1
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antibody
tgf
amino acid
seq
acid sequence
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PCT/JP2018/023920
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English (en)
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Yukikazu Natori
Wilber Huang
Hidekazu TOYOFUKU
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Bonac Corporation
Abnova (Taiwan) Corporation
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Publication of WO2018235964A1 publication Critical patent/WO2018235964A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity

Definitions

  • the present invention relates to a monoclonal antibody that specifically binds to an active transforming growth factor- ⁇ (TGF- ⁇ ) but does not bind to a latent TGF- ⁇ or off-target proteins, and a monoclonal antibody that specifically binds to latency associated peptide (LAP) and ⁇ a latent TGF- ⁇ but does not bind to an active TGF- ⁇ or off-target proteins.
  • TGF- ⁇ active transforming growth factor- ⁇
  • LAP latency associated peptide
  • TGF- ⁇ Transforming growth factor beta
  • TGF- ⁇ 1 to - ⁇ 5 multifunctional cytokine belonging to the transforming growth factor superfamily that includes five different isoforms (TGF- ⁇ 1 to - ⁇ 5) and many other signaling proteins produced by all white blood cell lineages.
  • the type 2 receptor kinase phosphorylates and activates the type 1 receptor kinase that activates a signaling cascade.
  • TGF- ⁇ also plays a crucial role in stem cell differentiation as well as T-cell regulation and differentiation. As such, it is a highly researched cytokine in the fields of cancer, auto-immune diseases, and infectious diseases .
  • TGF- ⁇ The peptide structures of the TGF- ⁇ isoforms are highly similar (homologies on the order of 70-80%). They are all encoded as large protein precursors; TGF- ⁇ contains 390 amino acids and TGF-p2 and TGF ⁇ 3 each contain 412 amino acids. They each have an N-terminal signal peptide of 20-30 amino acids that they require for secretion from a cell, a pro-region called LAP, and a 112-114 amino acid C-terminal region that becomes the mature TGF- ⁇ molecule following its release from the pro-region by proteolytic cleavage.
  • LAP pro-region
  • C-terminal region that becomes the mature TGF- ⁇ molecule following its release from the pro-region by proteolytic cleavage.
  • the TGF- ⁇ homodimer interacts with LAP, forming a complex called Small Latent Complex (SLC) .
  • SLC Small Latent Complex
  • This complex remains in the cell until it is bound by another protein called Latent TGF- -Binding Protein (LTBP) , forming a larger complex called Large Latent Complex (LLC) .
  • the LLC that gets secreted to the extracellular matrix (ECM) .
  • ECM extracellular matrix
  • the TGF- ⁇ precursor is cleaved from the propeptide but remains attached to it by noncovalent bonds.
  • ECM extracellular matrix
  • the TGF- ⁇ precursor is cleaved from the propeptide but remains attached to it by noncovalent bonds. After its secretion, it remains in ECM as an inactivated complex containing both LTBP and LAP which need to be further processed in order to release active TGF- ⁇ .
  • the attachment of TGF- ⁇ to the LTBP is by disulfide bond which allows it to remain inactive by preventing it from binding to its receptors.
  • TGF- ⁇ is known to be involved in the pathologies of various diseases including fibrosis such as pulmonary fibrosis and cancer.
  • an anti-TGF- ⁇ antibody that specifically recognizes an active TGF- ⁇ molecule is highly required.
  • almost of all known anti-TGF- ⁇ antibodies recognize not only an antive TGF- ⁇ but also a latent TGF- ⁇ and/or other off-target proteins such as TGF-p2, TGF-p3 and the like.
  • An object of the present invention is to provide a novel monoclonal antibody that specifically binds to an active TGF- ⁇ but does not bind to a latent TGF- ⁇ or off-target proteins.
  • An object of the present invention is to provide a novel monoclonal antibody that specifically binds to LAP and a latent TGF- ⁇ but does not bind to an active TGF- ⁇ or off-target proteins .
  • the present inventors have conducted intensive studies in an attempt to solve the aforementioned problems, predicted in silico evolutionally conserved unique amino acid sequences in LAP and mature TGF- ⁇ regions, immunized mice with peptides having said amino acid sequences derived from human TGF- ⁇ , fused antibody-producing cells obtained from mice with high titer with myeloma cells to give hybridomas, and selected hybridoma clones that produce an antibody specifically binding to an active TGF- ⁇ or a latent TGF ⁇ l/LAP by positive
  • the present inventors have conducted further studies to succeed in obtaining monoclonal antibody clones having
  • a monoclonal antibody that binds to an active TGF- ⁇ , and does not bind to a latent TGF- ⁇ comprising:
  • [3] The antibody of [1], wherein the CDR1, CDR2 and CDR3 contained in the heavy chain variable region are (a) , (b) and (c) above, respectively, and the CDR1, CDR2 and CDR3 contained in light chain variable region are (d) , (e) and (f) above, respectively.
  • [5] A monoclonal antibody that binds to an active TGF- ⁇ competitively with the antibody of any one of [1] to [4], and does not recognize a latent TGF- ⁇ .
  • a monoclonal antibody that binds to a latent TGF- ⁇ or LAP, and does not bind to an active TGF- ⁇ comprising:
  • the anti-TGF- ⁇ antibody of the present invention is highly specific to an active TGF- ⁇ or a latent TGF- ⁇ l/LAP, it can selectively detect its target molecule in a biological sample such as tissue specimen and body fluid.
  • Fig. 1 shows immunohistochemistry (IHC) staining of HCC tissue using 4 anti-active TGF- ⁇ antibodies.
  • Fig. 2 shows immunohistochemistry (IHC) staining of lung adenocarcinoma tissue using 4 anti-active TGF- ⁇ antibodies.
  • Fig. 3 shows immunohistochemistry (IHC) staining of lung adenocarcinoma tissue using 4 anti-active TGF- ⁇ antibodies.
  • Fig. 4 shows immunohistochemistry (IHC) staining of breast cancer tissue using 4 anti-active TGF- ⁇ antibodies.
  • Fig. 5 shows immunohistochemistry (IHC) staining of ovarian cancer tissue using 4 anti-active TGF- ⁇ antibodies.
  • Fig. 6 shows immunohistochemistry (IHC) staining of prostate cancer tissue using 4 anti-active TGF- ⁇ antibodies.
  • Fig. 7 shows immunohistochemistry (IHC) staining of HCC tissue using 4 anti-active TGF- ⁇ antibodies.
  • Fig. 8 shows immunohistochemistry (IHC) staining of 4 kinds of cancer tissues using anti-latent TGF-pi/LAP antibody 2F10.
  • Fig. 9 shows immunohistochemistry (IHC) staining of lungs of TGF-pi-Tg mice and wild type mice using anti-latent TGF- ⁇ /LAP antibody 2F10.
  • nucleotide sequence or amino acid sequence ed. Japan Patent Office
  • gene or “DNA” is used in the meaning that it includes not only a double-stranded DNA but also
  • the gene (DNA) in the specification includes, unless otherwise instructed, a double-stranded DNA including a human genomic DNA, a single-stranded DNA (plus strand) including a cDNA, a single- stranded DNA having a sequence complementary to the plus strand (complementary strand) and fragments thereof.
  • TGF- ⁇ gene means a human TGF- ⁇ gene (DNA) whose cDNA sequence is deposited to GenBank as accession No. NM_000660, or naturally occurring mutants or polymorphic variants thereof. Such mutants or polymorphic variants include, for example, those registered in the SNP database available form NCBI.
  • TGF- ⁇ protein or simply "TGF- ⁇ ” means a human TGF- ⁇ protein whose amino acid sequence is deposited to GenBank as accession No. NM_000651, wherein
  • position 1-29 is signal peptide
  • position 30-278 is LAP
  • position 279-390 is mature TGF- ⁇ , or a protein encoded by the naturally occurring mutant or polymorphic variant DNAs
  • the term “latent TGF- ⁇ ” means an inactive form of TGF- ⁇ consisting of a homodimer of mature TGF- ⁇ and a homodimer of LAP bound therewith via non-covalent bond, and optionally further LTBP.
  • active TGF- ⁇ means the
  • antibody used herein encompasses a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single- stranded antibody, or a part thereof capable of binding with its antigen such as an Fab fragment and the like.
  • epitope is a region of an antigen to which an antibody binds. In certain embodiments, it includes any site on an antigen that is capable of specific binding to an immunoglobulin.
  • Antigen determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge
  • an antibody specifically binds to its target antigen when it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules .
  • the basic structure of the antibody molecule is common to all classes, and constituted of a heavy chain having a
  • a heavy chain is generally composed of a polypeptide chain containing about 440 amino acids, has a characteristic structure for each class, and is called ⁇ , ⁇ , , ⁇ , ⁇ chain corresponding to IgG, IgM, IgA, IgD, IgE.
  • IgG includes IgGl, IgG2, IgG3, IgG4, which are called ⁇ , ⁇ 2, ⁇ 3, ⁇ 4, respectively.
  • a light chain is generally composed of a polypeptide chain containing about 220 amino acids, and two kinds of L type and K type, called ⁇ , chain, respectively, are known.
  • the peptide constitution of the basic structure of the antibody molecule contains two heavy chains and two light chains homologous to each other and bonded by a disulfide bond (S-S bond) and a noncovalent bond, and has a molecular weight of 150,000-190,000.
  • the two kinds of light chains can make a pair with any heavy chain.
  • Each antibody molecule is always composed of the same two light chains and the same two heavy chains .
  • a heavy chain contains 4 (5 in ⁇ , ⁇ chains) S-S bonds, and a light chain contains 2 S-S bonds, thereby forming one loop per 100-110 amino acid residues.
  • the steric structure is similar between loops, and called a structure unit or domain.
  • the domain present at the N terminal of heavy chain and light chain has a varying amino acid sequence even in a reference standard from the same class (subclass) of animals of the same species and is called a variable region (V region) (heavy chain variable region domain is indicated as V H , light chain variable region domain is indicated as V L ) .
  • V region variable region
  • V H variable region
  • V L variable region
  • the amino acid sequence on the C-terminal side is almost constant for each class or subclass and is called a constant region (C region) (each domain is indicated as C H 1, C H 2, C H 3 or C L ) .
  • An antigen determination site of an antibody is
  • variable regions of light chain and heavy chain are mostly limited to three small hypervariable regions present in both chains, and these regions are called complementarity determining region (CDR) .
  • CDR complementarity determining region
  • the part other than CDR is called a framework region (FR) , and is comparatively constant.
  • the framework region adopts ⁇ sheet conformation and CDR can form a loop that connects ⁇ sheet structures.
  • CDR in each chain is maintained in the tertiary structure thereof by a framework region and forms an antigen binding site together with CDR from other chain.
  • CDR of the antibody of the present invention is defined to be a CDR identified by analyzing the amino acid sequences of the variable regions (V H and V L ) of heavy chain and light chain of said antibody, by using publicly available CDR determination softwares (http://www.abysis.org and http : //www. ncbi . nlm.nih.gov/igblast/igblast . cgi) .
  • the CDRs in the heavy chain variable region are bounded by the residues at amino acid Nos . 26-32 (CDR1-H) , 52-57 (CDR2-H) and 99-109
  • CDR3-H of the amino acid sequence shown by SEQ ID NO: 7
  • the CDRs in the light chain variable region are bounded by the residues at amino acid Nos. 24-40 (CDR1-L) , 56-62 (CDR2-L) and 95-102 (CDR3-L) of the amino acid sequence shown by SEQ ID NO: 8.
  • the CDRs in the heavy chain variable region are bounded by the residues at amino acid Nos. 26-32 (CDR1-H) , 52-57 (CDR2-H) and 99-103 (CDR3-H) of the amino acid sequence shown by SEQ ID NO: 15, and the CDRs in the light chain variable region are bounded by the residues at amino acid Nos. 24-34 (CDR1-L), 50-56 (CDR2-L) and 89-97 (CDR3-L) of the amino acid sequence shown by SEQ ID NO: 16.
  • the portion other than CDRs of the variable region is called a framework region (FR) , and is relatively constant.
  • the framework region employs a ⁇ sheet conformation, and CDRs can form a loop connecting the ⁇ sheet structure.
  • CDRs in each chain are maintained in the three dimensional structure thereof by the framework regions and form an antigen binding site together with CDRs from the other chain.
  • Antibody binding can be confirmed by any known assay method, such as direct and indirect sandwich assays and
  • the binding of an anti-TGF- ⁇ antibody with an anctive TGF- ⁇ or a latent TGF ⁇ l/LAP polypeptide can be measured, for example, by the following method.
  • exemplified is a method comprising adsorbing a human TGF- ⁇ / ⁇ , ⁇ polypeptide (antigen) onto a solid phase, blocking the solid phase with a protein that is not involved in the subsequent antigen-antibody reaction or enzyme reaction (e.g., skim milk, albumin etc.), contacting and incubating a anti-TGF- ⁇ monoclonal antibody (test antibody) with the solid phase, removing an unreacted antibody by B/F separation, and adding a labeled secondary antibody
  • test antibody e.g., anti-human IgG, etc.
  • the solid phase for example, insoluble polysaccharides such as agarose, dextran and
  • cellulose synthetic resins such as plastic, polystylene, polyacrylamide and silicone (e.g., tube, microplate, etc.), or glass (beads, tube, etc.) can be used.
  • labeling agent radioisotopes, enzymes, fluorescent substances, luminescent substances and the like can be used.
  • radioisotopes [ 125 I] , [ 131 I] , [ 3 H] , [ 14 C] and the like can be mentioned.
  • the enzymes stable enzymes with high specific activity are preferred; for example, ⁇ - galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like can be mentioned.
  • fluorescent substances fluorescamine
  • luminescent substances examples include luminol, luminol
  • the competitive assays are carried out by adding a free antigen or known antibody to the reaction system of the solid phase and the test antibody in the binding assay using antigen-immobilized solid phase mentioned above. For example, a given concentration of test antibody solution and mixtures in which various concentrations of antigen are added to the test antibody solution are contacted and incubated with an antigen- immobilized solid phase, respectively, and the amounts of label on the respective solid phases are measured.
  • the binding constant can be calculated as the gradient of graph showing the results of Scatchard analysis based on the measured values for respective antigen concentrations.
  • an antibody binding to an active or latent TGF- ⁇ competitively with the antibody of the present invention can be identified by reacting a labeled known antibody (the inventive antibody) and various concentrations of test antibody with the antigen- immobilized solid phase, and selecting the test antibody that reduced the amount of label on the solid phase in a dose- dependent manner.
  • the present invention provides a monoclonal antibody that binds to an active TGF- ⁇ , and does not bind to a latent TGF- ⁇ (hereinafter sometimes to be referred to as “the anti-active TGF- ⁇ antibody of the present invention” or simply as “the antibody (1) of the present invention”) .
  • the present invention also provides a monoclonal antibody that binds to a latent TGF- ⁇ or LAP, and does not bind to an active TGF- ⁇ (hereinafter sometimes to be referred to as "the anti-latent TGF- ⁇ / ⁇ antibody of the present invention” or simply as “the antibody (2) of the present invention”) .
  • the antibody (1) of the present invention and the antibody (2) of the present invention are collectively referred to as "the anti-TGF- ⁇ antibody of the present invention” or simply as “the antibody of the present invention”.
  • the antibody of the present invention is further characterized by not recognizing any off-target
  • the antibody (1) of the present invention is a monoclonal antibody ( Ab) clone 2H4, 4D10 or an antibody that has the same complementarity
  • CDRs determining regions
  • the antibody (1) of the present invention is an antibody that binds to an active TGF- ⁇ competitively with the Mab 2H4 or 4D10, and does not recognize a latent TGF- ⁇ .
  • the antibody (2) of the present invention is a monoclonal antibody (MAb) clone 2F10 or an antibody that has the same complementarity determining regions (CDRs) as those of said monoclonal antibody.
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • the antibody (2) of the present invention is an antibody that binds to a latent TGF- ⁇ or LAP competitively with the Mab 2F10, and does not recognize an active TGF- ⁇ .
  • the antibody (1) of the present invention is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • CDR comprising the amino acid sequence shown by Lys Ser Ser Gin Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala (SEQ ID NO: 4) ,
  • CDR comprising the amino acid sequence shown by Gin Gin Ser Tyr His Leu Pro Thr (SEQ ID NO: 6), or
  • the binding property of an antibody can be determined by various binding assays described above.
  • an antibody containing the heavy chain and light chain variable regions of the above-mentioned (1) having one or more (e.g., 1, 2, 3, 4, 5 or 6) amino acid sequences selected from the amino acid sequences shown in SEQ ID NOs: 1 - 6, wherein one or two amino acid residues are substituted and/or deleted and/or added and/or inserted in each sequence, which
  • CDRs of (a) , (b) and (c) are set in this order from the N terminal of the heavy chain. That is, CDRs of (a) , (b) and (c) correspond to CDRl, CDR2 and CDR3 of the heavy chain, respectively.
  • CDRs of (d) , (e) and (f) are set in this order from the N terminal of the light chain. That is, CDRs of (d) , (e) and (f) correspond to CDRl, CDR2 and CDR3 of the light chain, respectively .
  • a still more preferable example of the antibody (1) of the present invention is
  • an antibody containing the heavy chain and light chain variable regions of the above-mentioned (1) having either one or both of SEQ ID NOs : 7 and 8, wherein one or more, preferably 1 - 20, more preferably 1 - 10, further preferably 1 - several (e.g., 1, 2, 3, 4 or 5), amino acid residues are substituted and/or deleted and/or added and/or inserted in each sequence, which specifically recognizes an active TGF- ⁇ but does not recognize a latent TGF- ⁇ .
  • the 50th residue is preferably Pro, and when the 11th residue is Arg, the 50th residue is preferably Leu.
  • the antibody (1) of the present invention can be an antibody that binds to an active TGF- ⁇ competitively with any of the above-mentioned anti-acitve TGF- ⁇ antibodies, and does not recognize a latent TGF- ⁇ .
  • Examples of such antibody include mouse anti-human active TGF- ⁇ antibody clones 6F12 and 7F10 described in the below- mentioned Examples.
  • the competitive binding of an antibody can be determined by competitive assays described above.
  • the antibody (2) of the present invention is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the binding property and competitive binding of an antibody can be determined by various binding assays described above .
  • an antibody containing the heavy chain and light chain variable regions of the above-mentioned (1) having one or more (e.g., 1, 2, 3, 4, 5 or 6) amino acid sequences selected from the amino acid sequences shown in SEQ ID NOs: 9 - 14, wherein one or two amino acid residues are substituted and/or deleted and/or added and/or inserted in each sequence, which
  • CDRs of (a) , (b) and (c) are set in this order from the N terminal of the heavy chain. That is, CDRs of (a) , (b) and (c) correspond to CDR1, CDR2 and CDR3 of the heavy chain, respectively.
  • CDRs of (d) , (e) and (f) are set in this order from the N terminal of the light chain. That is, CDRs of (d) , (e) and (f) correspond to CDR1, CDR2 and CDR3 of the light chain, respectively.
  • a still more preferable example of the antibody (2) of the present invention is
  • an antibody containing the heavy chain and light chain variable regions of the above-mentioned (1) having either one or both of SEQ ID NOs:15 and 16, wherein one or more,
  • amino acid residues are substituted and/or deleted and/or added and/or inserted in each sequence, which specifically recognizes a latent TGF- ⁇ or LAP but does not recognize an active TGF- ⁇ .
  • the antibody (2) of the present invention can be an antibody that binds to a latent TGF- ⁇ or LAP competitively with any of the above-mentioned anti-latent TGF- ⁇ l/LAP antibodies, and does not recognize an active TGF- ⁇ .
  • the competitive binding of an antibody can be determined by competitive assays described above.
  • invention is not particularly limited, it is preferably IgG, IgM or IgA, particularly preferably IgG.
  • the antibody of the present invention is not subject to limitation on the form of molecules as long as it has at least its CDRs for specifically recognizing and binding to the antigenic determinant (epitope) in addition to the whole antibody molecule, the antibody may, for example, be a
  • engineered conjugate molecule such as scFv, scFv-Fc, minibody, or diabody, or a derivative thereof modified with a molecule having protein stabilizing action, such as polyethylene glycol (PEG), or the like, and the like.
  • PEG polyethylene glycol
  • the antibody of the present invention can be produced by a method of antibody production known per se. Hereinafter, a method of preparing an immunogen for producing the antibody of the present invention, and a method of producing the antibody are described.
  • the antigen used to prepare the antibody of the present invention may be the whole latent or mature TGF- ⁇ or the whole LAP polypeptide or a partial peptide thereof, a (synthetic) peptide having one or more kinds of the same antigen
  • polypeptide that consists of 6 to 15 amino acids is used as an immunogen. More preferably, evolutionally conserved unique amino acid sequences in LAP and mature TGF- ⁇ regions,
  • Such evolutionally conserved unique amino acid sequences can be predicted in silico using a commercially available epitope prediction software.
  • the whole latent or mature TGF- ⁇ or LAP polypeptide or a partial peptide thereof is produced by, for example, (a) preparing the same from a human tissue or cells, by a method known to the public or its modified method, (b) chemically synthesizing the same by a publicly known method of peptide synthesis using a peptide synthesizer and the like, (c) culturing a transformant comprising a DNA that encodes the whole polypeptide or a partial peptide thereof, or (d)
  • oligopeptide can be linked with a suitable carrier protein such as keyhole limpet hemocyanin (KLH) to confer KLH.
  • KLH keyhole limpet hemocyanin
  • the immunogen prepared as mentioned above is administered as is, or along . with a carrier or a diluent, to a warm-blooded animal at a site enabling antibody production by the methods such as intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection and the like.
  • Freund' s complete adjuvant or Freund' s incomplete adjuvant may be administered. Dosing is normally performed about 2 to 10 times in total every 1 to 6 weeks.
  • mouse, rat rabbit, goat, monkey, dog, guinea pig, sheep, donkey and chicken preferably mouse, rat and rabbit can be mentioned.
  • the immunogen can be subjected to in vitro immunization method.
  • animal cells used in the in vitro immunization method lymphocytes, preferably B-lymphocytes and the like, isolated from peripheral blood, spleen, lymph node and the like of a human and the above-described warm-blooded animals (preferably mouse or rat) can be mentioned.
  • lymphocytes preferably B-lymphocytes and the like, isolated from peripheral blood, spleen, lymph node and the like of a human and the above-described warm-blooded animals (preferably mouse or rat) can be mentioned.
  • the spleen is extirpated from an about 4- to 12-week-old animal, and
  • splenocytes are separated and rinsed with a appropriate medium [e.g., Dulbecco's modified Eagle medium (DMEM) , RP I1640 medium, Ham's F12 medium and the like], after which the splenocytes are suspended in an antigen-containing medium supplemented with fetal calf serum (FCS; about 5 to 20%) and cultured using a C0 2 incubator and the like for about 4 to 10 days.
  • DMEM Dulbecco's modified Eagle medium
  • FCS fetal calf serum
  • FCS fetal calf serum
  • the antigen concentration include, but are not limited to, 0.05 - 5 ⁇ g. It is preferable to prepare a culture supernatant of thymocytes of an animal of the same strain (preferably at about 1 to 2 weeks of age) according to a conventional method, and to add the supernatant to the medium.
  • cytokines such as IL-2, IL-4 IL-5, and IL-6 and the like, and if necessary, an adjuvant substance (e.g., muramyldipeptide and the like) along with the antigen.
  • an adjuvant substance e.g., muramyldipeptide and the like
  • an antibody-producing hybridoma by selecting an individual or cell population showing an elevated antibody titer from among antigen-immunized warm-blooded animals (e.g., mice, rats) or animal cells (e.g., human, mouse, rat),
  • antigen-immunized warm-blooded animals e.g., mice, rats
  • animal cells e.g., human, mouse, rat
  • a measurement of serum antibody titer can be performed by, for example, reacting a labeled antigen and an antiserum, and thereafter determining the activity of the label bound to the antibody.
  • the myeloma cells are not subject to limitation, as long as they are capable of producing a hybridoma that secretes a large amount of antibody, those that do not produce or secrete the antibody per se are preferable, with greater preference given to those of high cell fusion efficiency.
  • HAT hyperxanthine, aminopterin, thymidine
  • mice myeloma cells As examples of the mouse myeloma cells, NS-1, P3U1, SP2/0, AP-1 and the like can be mentioned; as examples of the rat myeloma cells, R210.RCY3, Y3-Ag 1.2.3 and the like can be mentioned; as examples of the human myeloma cells, SKO-007, GM 1500-6TG-2, LICR-L0N-HMy2, UC729-6 and the like can be mentioned;
  • Fusion operation can be performed according to a known method, for example, the method of Koehler and ilstein [Nature, 256, 495 (1975)].
  • a fusion promoter polyethylene glycol (PEG) , Sendai virus and the like can be mentioned, and PEG and the like are preferably used.
  • PEG1000 to PEG6000 which are of low toxicity and relatively low viscosity, are preferable.
  • PEG concentration about 10 to 80%,
  • DMSO e.g., about 10 to 20%
  • pH of the fusion solution about 4 to 10, preferably about 6 to 8 can be mentioned.
  • lymphocytes and myeloma cells
  • incubation normally at 20 to 40°C, preferably at 30 to 37°C, normally for 1 to 10 minutes.
  • An antibody-producing cell line can also be obtained by infecting antibody-producing cells with a virus capable of transforming lymphocytes to immortalize the cells.
  • viruses for example, Epstein-Barr (EB) virus and the like can be mentioned.
  • EB Epstein-Barr
  • the majority of persons have immunity because they have ever been infected with this virus in an asymptomatic infection of infectious mononucleosis, virion is also produced when the ordinary EB virus is used; therefore, appropriate purification must be performed.
  • a recombinant EB virus that retains the capability of immortalizing B lymphocytes but lacks the
  • An antibody-producing B cell line can be obtained by, for example, culturing these cells using a medium supplemented with serum and penicillin/streptomycin (P/S) (e.g., RPMI1640) or a serum-free medium supplemented with a cell growth factor, thereafter separating the culture supernatant by filtration or centrifugation and the like, suspending therein antibody-producing B lymphocytes at a suitable concentration (e.g., about 10 7 cells/mL) , and incubating the suspension normally at 20 to 40°C, preferably at 30 to 37°C, normally for about 0.5 to 2 hours.
  • P/S penicillin/streptomycin
  • a suitable concentration e.g., about 10 7 cells/mL
  • T lymphocytes When human antibody-producing cells are provided as mixed lymphocytes, it is preferable to previously remove T lymphocytes by allowing them to form an E rosette with, for example, sheep erythrocytes and the like, to increase transformation frequency of EB virus, because the majority of persons have T lymphocytes which exhibit cytotoxicity to cells infected with EB virus. It is also possible to select
  • lymphocytes specific for the target antigen by mixing sheep erythrocytes, previously bound to a soluble antigen, with antibody-producing B lymphocytes, and separating the rosette using a density gradient of percoll and the like. Furthermore, because antigen-specific B lymphocytes are capped by adding the antigen in large excess so that they no longer present IgG to the surface, mixing with sheep erythrocytes bound to anti-IgG antibody results in the formation of rosette only by antigen- nonspecific B lymphocytes. Therefore, by collecting a layer of cells that don't form rosette from this mixture using a density gradient of percoll and the like, it is possible to select antigen-specific B lymphocytes.
  • Human antibody-secreting cells having acquired the capability of proliferating indefinitely by the transformation can be back fused with mouse or human myeloma cells in order to stably sustain the antibody-secreting ability.
  • myeloma cells the same as those described above can be used.
  • Hybridoma screening and breeding are normally performed using a medium for animal cells (e.g., RPMI1640) containing 5 to 20% FCS or a serum-free medium supplemented with cell growth factors, with the addition of HAT (hypoxanthine, aminopterin, thymidine) .
  • HAT hypoxanthine, aminopterin, thymidine
  • concentrations of hypoxanthine, aminopterin and thymidine about 0.1 mM, about 0.4 ⁇ and about 0.016 mM and the like, respectively.
  • ouabain resistance can be used. Because human cell lines are more susceptible to ouabain than mouse cell lines, it is possible to eliminate unfused human cells by adding ouabain at about 10 "7 to 10 ⁇ 3 M to the medium.
  • feeder cells In selecting a hybridoma, it is preferable to use feeder cells or culture supernatants of certain cells.
  • the feeder cells an allogenic cell species having a lifetime limited so that it dies after helping the emergence of hybridoma, cells capable of producing large amounts of a growth factor useful for the emergence of hybridoma with their proliferation potency reduced by irradiation and the like, and the like are used.
  • mouse feeder cells splenocytes, macrophage, blood, thymocytes and the like can be mentioned; as the human feeder cells, peripheral blood mononuclear cells and the like can be mentioned.
  • cell culture supernatant primary culture supernatants of the above-described various cells and culture supernatants of various established cell lines can be mentioned.
  • a hybridoma can also be selected by reacting a fluorescein-labeled antigen with fusion cells, and thereafter separating the cells that bind to the antigen using a fluorescein-labeled antigen with fusion cells, and thereafter separating the cells that bind to the antigen using a fluorescein-labeled antigen with fusion cells, and thereafter separating the cells that bind to the antigen using a fluorescein-labeled antigen with fusion cells, and thereafter separating the cells that bind to the antigen using a
  • FACS fluorescence-activated cell sorter
  • aminopterin can be removed 2 weeks after fusion and beyond because most myeloma cells die within 10 to 14 days.
  • a human hybridoma is normally maintained in a medium supplemented with aminopterin for about 4 to 6 weeks after fusion.
  • hypoxanthine and thymidine be removed more than one week after the removal of aminopterin. That is, in the case of mouse cells, for example, a complete medium (e.g., RPMI1640 supplemented with 10% FCS) supplemented with hypoxanthine and thymidine (HT) is added or exchanged 7 to 10 days after fusion. About 8 to 14 days after fusion, visible clones emerge. Provided that the diameter of clone has reached about 1 mm, the amount of antibody in the culture supernatant can be measured.
  • the measurement of the amount of antibody can be performed by, for example, a method comprising adding the hybridoma culture supernatant to a solid phase (e.g.,
  • an anti-immunoglobulin (IgG) antibody an antibody against IgG derived from an animal of the same species as the animal from which the original antibody-producing cells are derived is used
  • protein A which had been labeled with a radioactive substance (e.g., 125 I, 131 I, 3 H, 1 C)
  • enzyme e.g., ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase,
  • fluorescent substance e.g., fluorescamine, fluorescein isothiocyanate
  • luminescent substance e.g., fluorescamine, fluorescein isothiocyanate
  • a method comprising adding the hybridoma culture supernatant to a solid phase to which an anti-IgG antibody or protein A is adsorbed, adding the target antigen or derivatives thereof or partial peptide thereof labeled with the same labeling reagent as described above, and detecting the antibody against the target antigen bound to the solid phase and the like.
  • Cloning by limiting dilution can be performed by, for example, the following
  • the amount of antibody is measured as described above, and positive wells are selected. Selected suitable feeder cells are previously added to a 96-well plate. Cells are collected from the antibody-positive wells and suspended in complete medium (e.g., RMPI1640 supplemented with 10% FCS and P/S) to obtain a density of 30 cells/mL; 0.1 mL (3 cells/well) of this suspension is added to the well plate with feeder cells added thereto; a portion of the remaining cell suspension is diluted to 10 cells/mL and sown to other wells (1 cell/well) in the same way; the still remaining cell suspension is diluted to 3 cells/mL and sown to other wells (0.3 cells/well).
  • complete medium e.g., RMPI1640 supplemented with 10% FCS and P/S
  • the cells are cultured for about 2 to 3 weeks until a visible clone appears, when the amount of antibody is measured to select positive wells, and the selected cells are recloned.
  • cloning is relatively difficult, so that a plate in which cells are seeded at 10 cells/well is also prepared.
  • a monoclonal antibody-producing hybridoma can be obtained normally by two times of subcloning, it is desirable to repeat recloning regularly for several more months to confirm the stability thereof.
  • the hybridomas producing a monoclonal antibody against an active TGF- ⁇ obtained as described above are then subjected to the second screening.
  • the second screening not only an active TGF- ⁇ and/or a partial peptide thereof used as
  • the hybridomas producing a monoclonal antibody against a latent TGF- ⁇ / ⁇ obtained as described above are also subjected to the second screening.
  • the second screening not only a latent TGF- ⁇ and/or a partial peptide of LAP used as immunogen, but also an active TGF- ⁇ can also be used as the probe.
  • an active TGF- ⁇ can also be used as the probe.
  • TGF- ⁇ and/or a partial peptide thereof but not with a latent TGF- ⁇ can be selected as a hybridoma producing an anti-active TGF- ⁇ antibody of the present invention.
  • monoclonal antibody that reacted with a latent TGF- ⁇ and/or a partial peptide of LAP but not with an active TGF- ⁇ can be selected as a hybridoma producing an anti-latent TGF ⁇ l/LAP antibody of the present invention.
  • the antibody of the present invention does not cross-react any off-target proteins
  • the hybridomas are predicted by the amino acid sequence of its epitope, including TGF- 2, TGF-P3 and the like. Therefore, the hybridomas
  • Hybridomas thus obtained can be cultured in vitro or in vivo.
  • a method of in vitro culture a method comprising gradually scaling up a monoclonal antibody-producing hybridoma obtained as described above, from a well plate, while keeping the cell density at, for example, about 10 5 to 10 6 cells/mL, and gradually lowering the FCS concentration, can be mentioned.
  • a method of in vivo culture for example, a method of in vivo culture
  • Separation and purification of the monoclonal antibody are performed according to a method known per se, for example, a method of immunoglobulin separation and purification [e.g., salting-out, alcohol precipitation, isoelectric point
  • a monoclonal antibody can be produced by culturing a hybridoma in or outside the living body of a warm-blooded animal, and harvesting the antibody from the body fluid or culture thereof.
  • Examples of the anti-active TGF- ⁇ antibody of the present invention obtained as mentioned above include mouse anti-human active TGF- ⁇ antibody clones 2H4, 4D10, 6F12 and 7F10 described in the below-mentioned Examples.
  • Examples of the anti-latent TGF- ⁇ / ⁇ antibody of the present invention obtained as mentioned above include mouse anti-human latent
  • TGF- i/LAP antibody clone 2F10 described in the below-mentioned Examples. As a result of amino acid sequence determination, it is revealed that 2H4 or 4D10 antibody has a heavy chain
  • 2F10 antibody has a heavy chain containing a variable region consisting of the amino acid sequence shown by SEQ ID NO: 15 and a light chain containing a variable region consisting of the amino acid sequence shown by SEQ ID NO: 16.
  • cDNAs that encode the heavy chain and light chain of an anti-active TGF- ⁇ or a latent TGF ⁇ l/LAP antibody thus obtained can be isolated from cDNA library derived from a hybridoma producing the antibody and cloned into appropriate expression vector (s) functional in a host cell of interest by conventional methods. Then, a host cell is
  • Useful host cells include animal cells, for example, mouse myeloma cells as described above, as well as Chinese hamster ovary (CHO) cells, monkey-derived COS-7 cells, Vero cells, rat-derived GHS cells and the like.
  • animal cells for example, mouse myeloma cells as described above, as well as Chinese hamster ovary (CHO) cells, monkey-derived COS-7 cells, Vero cells, rat-derived GHS cells and the like.
  • the host cell is cultured in a suitable medium for a given period, the conditioned medium is recovered, and the antibody protein is purified by a conventional method, whereby the antibody of the present invention can be isolated.
  • transgenic plant prepared by microinjection or electroporation for protoplast, the particle gun method, the Ti vector method and the like for intact cells, using as the host cell a cell of a plant for which transgenic technology has been established, and which is cultured in large amounts as a major crop, such as corn, rice, wheat, soybean or tobacco.
  • the antibody of the present invention is capable of specifically recognizing either an active TGF- ⁇ or a latent TGF- i/LAP, and does not cross-react with any off-target proteins, it can be used for precise detection and quantitation of an active TGF- ⁇ or a latent TGF- ⁇ / ⁇ , ⁇ in a test cell sample.
  • the whole antibody molecule may be used, and any fragment thereof, such as the F(ab')2 ⁇ Fab' or Fab fraction of the antibody molecule, may also be used.
  • the measurement method using the antibody of the present invention should not be particularly limited, and any measurement method can be used.
  • a labeling agent to be used for the measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like can be used.
  • a radioisotope for example, [ 125 I] , [ 131 I], [ 3 H] , [ 1 C] and the like can be used.
  • the above- described enzyme is preferably stable and has a high specific activity and, for example, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like can be used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiocyanate (FITC) , phycoerythrin (PE) and the like can be used.
  • luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like can be used.
  • the antibody of the present invention may be directly or indirectly labeled with a labeling agent.
  • the anti-TGF- ⁇ antibody is an unlabeled antibody and an active TGF- ⁇ or latent TGF ⁇ l/LAP can be detected by the labeled second antibody such as anti-serum or anti-Ig antibody against the animal from which the anti-TGF- ⁇ antibody was produced.
  • the biotinylated second antibody can be used and a conjugate of TGF ⁇ l-the antibody of the present invention-the second antibody can be formed and
  • test cell sample can be fixed and
  • the cells reacted with the anti- TGF- ⁇ antibody can be visualized with the labeled second antibody and analyzed using a confocal laser scanning microscope, a flexible automated cell imaging system IN Cell Analyzer (Amarsham/GE) and the like.
  • the immunogens thus obtained were emulsified with an equal volume of Complete Freund' s Complete Adjuvant (CFA) or Incomplete Freund' s Adjuvant (IFA) and injected into mice intraperitoneally. Thereafter, booster injection was performed, and the mice were sacrificed.
  • CFA Complete Freund' s Complete Adjuvant
  • IFA Incomplete Freund' s Adjuvant
  • Lymphocytes from the spleen of the immunized mice with high titer by indirect ELISA of sera were fused with mouse myeloma cells using polyethylene glycol. Fused cells were seeded in 96-well tissue culture plates, and hybridoma (64 clones) were selected by adding hybridoma medium.
  • TGF- ⁇ overexpressing cell or tissue lysate was loaded onto SDS-polyacrylamide gel, reacted with 5x diluted hybridoma sup and iitimunoreactivity was visualized using HRP- conjugated goat anti-mouse IgG. Ten out of 11 clones were positive to both cell and tissue lysates.
  • IF immunofluorescence
  • Isotype of monoclonal antibody was analyzed by using mouse monoclonal antibody isotyping test kit. 2H4, 4D10, 6F12 and 7F10 antibodies belonged to IgGl isotype.
  • an anti-latent TGF-pi/LAP monoclonal antibody clone (2F10) was obtained by an antigen peptide from LAP region-immunized mice.
  • IHC immunohistochemistry
  • 2H4, 4D10 and 2F10 antibodies were purified from each hibridoma producing the same, and the amino acid sequences of heavy and light chain variable regions by a conventional method. As a result, the amino acid sequences of the hevy and light chain variable regions of these antibodies were as shown below.
  • PTFGGGTKLE IKR (SEQ ID -NO: 8; wherein the 11th and 50th Xaas stand for Leu and Pro, respectively) [4D10 heavy chain variable region]
  • PTFGGGTKLE IKR (SEQ ID NO: 8; wherein the 11th and 50th Xaas stand for Arg and Leu, respectively) [2F10 heavy chain variable region]
  • CDRs of the heavy and light chains of 2H4, 4D10 and 2F10 antibodies were determined.
  • the boxed sequences in each of SEQ ID Nos:7, 8, 15 and 16 are CDR1, CDR2 and CDR3 from N-terminus in this order.
  • the antibody of the present invention is highly useful for detecting only either an active TGF- ⁇ or a latent TGF- ⁇ /LAP in a biological sample such as tissue specimen and body fluid.

Abstract

La présente invention concerne un anticorps monoclonal qui reconnaît spécifiquement un TGF-β1 actif et ne reconnaît pas un TGF-β1 latent. L'invention concerne également un anticorps monoclonal qui reconnaît spécifiquement un TGF-β1 ou LAP latent et ne reconnaît pas un TGF-β1 actif. Ces anticorps sont en outre caractérisés par une non-réactivité croisée avec des protéines hors cibles.
PCT/JP2018/023920 2017-06-19 2018-06-19 Anticorps anti-tgf-bêeta1 WO2018235964A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11312767B2 (en) 2019-08-28 2022-04-26 Chugai Seiyaku Kabushiki Kaisha Cross-species anti-latent TGF-beta 1 antibodies

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